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<!-- NOTEX -->
Auxiliary version for preparing course notes

* [[#Kontakt]]
* [[#Úvod]]
* [[#Pravidlá]]

{|
|-
| 2019-02-21 || (BB) Introduction to Perl [[#Lperl|Lecture]], [[#HWperl|Homework]]
|-
| 2019-02-28 || (BB) Command-line tools, Perl one-liners [[#Lbash|Lecture]], [[#HWbash|Homework]]
|-
| 2019-03-07 || (BB) Job scheduling and make [[#Lmake|Lecture]], [[#HWmake|Homework]]
|-
| 2019-03-14 || (BB) Python and SQL for beginners [[#Lpython|Lecture]], [[#HWpython|Homework]]
|-
| 2019-03-21 || (VB) Web crawling, HTML parsing in Python [[#Lweb|Lecture INF]], [[#HWweb|Homework INF]]
|-
| || (BB) Bioinformatics 1 (genome assembly) [[#Lbioinf1|Lecture 5 bin]], [[#HWbioinf1|Homework 5 bin]]
|-
| 2019-03-28 || (VB) Text data processing, flask [[#Lflask|Lecture 6 inf]], [[#HWflask|Homework 6 inf]] 
|-
| || (BB) Bioinformatics 2 (gene finding, RNA-seq) [[#L06bin|Lecture 6 bin]], [[#HW06bin|Homework 6 bin]]
|-
| 2019-04-04 || (VB) Data visualization in JavaScript [[#Ljavascript|Lecture 7 inf]], [[#HWjavascript|Homework inf]]
|-
| || (BB) Bioinformatics 3 (polymorphisms) [[#L07bin|Lecture 7 bin]], [[#HW07bin|Homework 7 bin]]
|-
| 2019-04-11 || (BB) R, part 1 [[#L08|Lecture 8]], [[#HW08|Homework 8]]
|-
| 2019-04-18 || Easter (project proposals due Wednesday April 17)
|-
| 2019-04-25 || (BB) no lecture
|-
| 2019-05-02 || (BB) R, part 2 [[#L09|Lecture 9]], [[#HW09|Homework 9]]
|-
| 2019-05-09 || (VB) Cloud computing [[#L10|Lecture 10]], [[#HW10|Homework 10]]
|-
| 2019-05-16 || no lecture
|}
=Kontakt=
'''Vyučujúci'''

* [http://compbio.fmph.uniba.sk/~bbrejova/ doc. Mgr. Broňa Brejová, PhD.]  miestnosť M-163 <!-- , [[Image:e-bb.png]] -->
* [http://compbio.fmph.uniba.sk/~tvinar/ Mgr. Tomáš Vinař, PhD.], miestnosť M-163 <!-- , [[Image:e-tv.png]] -->
* [http://dai.fmph.uniba.sk/w/Vladimir_Boza/sk Mgr. Vladimír Boža, PhD.], miestnosť M-25 <!-- , [[Image:e-vb.png]] -->
<!-- * [http://dai.fmph.uniba.sk/~siska/ RNDr. Jozef Šiška, PhD.], miestnosť I-7 -->
* Konzultácie po dohode emailom

'''Rozvrh'''
*  Štvrtok 15:40-18:00 M-217
<!-- /NOTEX -->

=Introduction=
==Target audience==
This course is offered  at the Faculty of Matematics, Physics and Informatics, Comenius University in Bratislava for the students of the second year of the bachelor Bionformatics study program and the students of the bachelor and master Computer Science study programs. It is a prerequisite of the master-level state exams in Bioinformatics and Machine Learning. However, the course is open to students from other study programs if they satisfy the following informal prerequisites. 

We assume that the students are proficient in programming in at least one programming language and are not afraid to learn new languages. We also assume basic knowledge of work on the Linux command-line (at least basic commands for working with files and folders, such as cd, mkdir, cp, mv, rm, chmod). Although most technologies covered in this course can be used for processing data from many apication areas, we will illustrate some of them on examples from bioinformatics. We will explain necessary terminology from biology as needed.

The basic use of command-line tools can be learned for example by using [http://korflab.ucdavis.edu/bootcamp.html a tutorial by Ian Korf]. 

==Course objectives==

Computer science courses cover many interesting algorithms, models and methods that can used for data analysis. However, when you want to use these methods for real data, you will typically need to make considerable efforts to obtain the data, pre-process it into a suitable form, test and compare different methods or settings, and arrange the final results in informative tables and graphs. Often, these activities need to be repeated for different inputs, different settings, and so on. For example in bioinformatics, it is possible to find a job where your main task will be data processing using existing tools, possibly supplemented by small custom scripts. This course will cover some programming languages and technologies suitable for these activities.

This course is particularly recommended for students whose bachelor or master thesis involves substantial empirical experiments (e.g. experimental evaluation of your methods and comparison with other methods on real or simulated data). 

==Basic guidelines for working with data ==

As you know, in programming it is recommended to adhere to certain practices, such as good coding style, modular desgn, thorough testing etc. Such practices add a little extra work, but are much more efficient in the long run. Similar good practices exist for data analysis. As an introduction we recommend the following article by a well-known bionformatician William Stafford Noble (his advice applies outside of bionformatics as well):
* Noble WS. [http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1000424 A quick guide to organizing computational biology projects.] PLoS Comput Biol. 2009 Jul 31;5(7):e1000424.

Several important recommendations:
*  Noble 2009: '''"Everything you do, you will probably have to do over again."'''
** After doing an entire analysis, you often find out that there was a problem with the input data or one of the early steps and therefore everything needs to be redone
** Therefore it is better to use techniques that allow you to keep all detailes of your workflow and to repeat them if needed
** Try to avoid manually changing files, because this makes reruning analyses harder and more error-prone

* '''Document all steps of your analysis'''
** Note what have you done, why have you done it, what was the result
** Some of these things may seem obvious to you at present, but you may forgot them in a few weeks or months and you may need them to write up your thesis or to repeat the analysis
** Good documentation is also indispensable for collaorative projects


* '''Keep a logical structure of your files and folders'''
** Their names should be indicative of the contents (create a sensible naming scheme)
** However, if you have too many versions of the experiment, it may be easier to name them by date rather than create new long names (your notes should then detail the meaning of each dated version)

* '''Try to detect problems in the data'''
** Often big files may hide some problems in the format, unexpected values etc.  These may confuse your programs and make the results meaningless
** In your scripts, check that the input data conform to your expectations (format, values in reasonable ranges etc)
** In unexpected circumstances, scripts should terminate with an error message and a non-zero exit code
** If your script executes another program, check its exit code
** Also check intermedate results as often as possible (by manual inspection, computing various statistics etc) to detect errors in the data and your code

<!-- TEX
==Software requirements and the accompanying data==
* In this course, the students are given access to a Linux server with all necessary tools installed
* All the tools are freely available and most of them can be easily installed e.g. as Ubuntu packages
* The server also contains data needed for the exercises, but this data can be also obtained from an accompanying website.
* In the text below, replace /tasks/ with the path to your copy of the accompanying data
/TEX -->

<!-- NOTEX -->
=Pravidlá=
==Známkovanie==

* Domáce úlohy: 55%
* Návrh projektu: 5%
* Projekt: 40% 

Stupnica:
* A: 90 a viac, B:80...89, C: 70...79, D: 60...69, E: 50...59,  FX: menej ako 50%

==Formát predmetu==
* Každý týždeň 3 vyučovacie hodiny, z toho cca prvá je prednáška a ďalšie dve cvičenia. Na cvičeniach samostatne riešite príklady, ktoré doma dokončíte ako domácu úlohu.
* Niektoré týždne bude zvlášť úloha pre študentov bakalárskeho programu Bioinformatika a zvlášť pre ostatných. Ak by ste chceli riešiť iné zadanie, než je pre vás určené, musíte získať vopred súhlas vyučujúcich.
* Cez skúškové obdobie budete odovzdávať projekt. Po odovzdaní projektov sa bude konať ešte diskusia o projekte s vyučujúcimi, ktorá môže ovplyvniť vaše body z projektu.
* Budete mať konto na Linuxovom serveri určenom pre tento predmet. Toto konto používajte len na účely tohto predmetu a snažte sa server príliš svojou aktivitou nepreťažiť, aby slúžil všetkým študentom. Akékoľvek pokusy úmyselne narušiť chod servera budú považované za vážne porušenie pravidiel predmetu.

==Domáce úlohy==
* Termín DÚ týkajúcej sa aktuálnej prednášky je vždy do 9:00 v deň nasledujúcej prednášky (t.j. väčšinou o necelý týždeň od zadania).
* Domácu úlohu odporúčame začať robiť na cvičení, kde vám môžeme prípadne poradiť. Ak máte otázky neskôr, pýtajte sa vyučujúcich emailom.
* Domácu úlohu môžete robiť na ľubovoľnom počítači, pokiaľ možno pod Linuxom. Odovzdaný kód alebo príkazy by však mali byť spustiteľné na serveri pre tento predmet, nepoužívajte teda špeciálny softvér alebo nastavenia vášho počítača.
* Domáca úloha sa odovzdáva nakopírovaním požadovaných súborov do požadovaného adresára na serveri. Konkrétne požiadavky budú spresnené v zadaní.
* Ak sú mená súborov špecifikované v zadaní, dodržujte ich. Ak ich vymýšľate sami, nazvite ich rozumne. V prípade potreby si spravte aj podadresáre, napr. na jednotlivé príklady.
* Dbajte na prehľadnosť odovzdaného zdrojového kódu (odsadzovanie, rozumné názvy premenných, podľa potreby komentáre)

===Protokoly===
* Väčšinou bude požadovanou súčasťou úlohy textový dokument nazvaný protokol.
* Protokol môže byť vo formáte .txt alebo .pdf a jeho meno má byť '''protocol.pdf''' alebo '''protocol.txt''' (nakopírujte ho do odovzdaného adresára)
* Protokol môže byť po slovensky alebo po anglicky.
* V prípade použitia txt formátu a diakritiky ju kódujte v UTF8, ale pre jednoduchosť môžete protokoly písať aj bez diakritiky. Ak je protocol v pdf formáte, mali by sa v ňom dať selektovať texty. 
* Vo väčšine úloh dostanete kostru protokolu, dodržujte ju.

'''Hlavička protokolu, vyhodnotenie'''
* Na vrchu protokolu uveďte meno, číslo domácej úluhy a vaše vyhodnotenie toho, ako sa vám úlohu podarilo vyriešiť. Vyhodnotenie je prehľadný zoznam všetkých príkladov zo zadania, ktoré ste aspoň začali riešiť a kódov označujúcich ich stupeň dokončenia:
** kód HOTOVO uveďte, ak si myslíte, že tento príklad máte úplne a správne vyriešený
** kód ČASŤ uveďte, ak ste nevyriešili príklad celý a do poznámky za kód stručne uveďte, čo máte hotové a čo nie, prípadne ktorými časťami si nie ste istí.
** kód MOŽNO uveďte, ak príklad máte celý, ale nie ste si istí, či správne. Opäť v poznámke uveďte, čím si nie ste istí.
** kód NIČ uveďte, ak ste príklad ani nezačali riešiť
* Vaše vyhodnotenie je pre nás pomôckou pri bodovaní. Príklady označené HOTOVO budeme kontrolovať námatkovo, k príkladom označeným MOŽNO sa vám pokúsime dať nejakú spätnú väzbu, takisto aj k príkladom označeným ČASŤ, kde v poznámke vyjadríte, že ste mali nejaké problémy.
* Pri vyhodnotení sa pokúste čo najlepšie posúdiť správnosť vašich riešení, pričom kvalita vášho seba-hodnotenia môže vplývať na celkový počet bodov.

'''Obsah protokolu'''
* Ak nie je v zadaní určené inak, protokol by mal obsahovať nasledovné údaje:
** '''Zoznam odovzdaných súborov:''' o každom súbore uveďte jeho význam a či ste ho vyrobili ručne, získali z externých zdrojov alebo vypočítali nejakým programom. Ak máte väčšie množstvo súborov so systematickým pomenovaním, stačí vysvetliť schému názvov všeobecne. Súbory, ktorých mená sú špecifikované v zadaní, nemusíte v zozname uvádzať.
** '''Postupnosť všetkých spustených príkazov,''' prípadne iných krokov, ktorými ste dospeli k získaným výsledkom. Tu uvádzajte príkazy na spracovanie dát a spúšťanie vašich či iných programov. Netreba uvádzať príkazy súvisiace so samotným programovaním (spúšťanie editora, nastavenie práv na spustenie a pod.), s kopírovaním úlohy na server a pod. Uveďte aj stručné '''komentáre''', čo bolo účelom určitého príkazu alebo skupiny príkazov.
** '''Zoznam zdrojov:''' webstránky a pod., ktoré ste pri riešení úlohy použili. Nemusíte uvádzať webstránku predmetu a zdroje odporučené priamo v zadaní.
Celkovo by protokol mal umožniť čitateľovi zorientovať sa vo vašich súboroch a tiež v prípade záujmu vykonať rovnaké výpočty, akými ste dospeli vy k výsledku. Nemusíte písať slohy, stačia zrozumiteľné a prehľadné heslovité poznámky.

==Projekty==

Cieľom projektu je vyskúšať si naučené zručnosti na konkrétnom projekte spracovania dát. Vašou úlohou je zohnať si dáta, tieto dáta analyzovať niektorými technikami z prednášok, prípadne aj inými technológiami a získané výsledky zobraziť v prehľadných grafoch a tabuľkách. Ideálne je, ak sa vám podarí prísť k zaujímavým alebo užitočným záverom, ale hodnotiť budeme hlavne voľbu vhodného postupu a jeho technickú náročnosť. Rozsah samotného programovania alebo analýzy dát by mal zodpovedať zhruba trom domácim úlohám, ale celkovo bude projekt náročnejší, lebo na rozdiel od úloh nemáte postup a dáta vopred určené, ale musíte si ich vymyslieť sami a nie vždy sa prvý nápad ukáže ako správny. V projekte môžete využiť aj existujúce nástroje a knižnice, ale pokiaľ možno používajte nástroje spúšťané na príkazovom riadku. 

Zhruba v dvoch tretinách semestra budete odovzdávať '''návrh projektu''' (formát txt alebo pdf, rozsah 0.5-1 strana). V tomto návrhu uveďte, aké dáta budete spracovávať, ako ich zoženiete, čo je cieľom analýzy a aké technológie plánujete použiť. Ciele a technológie môžete počas práce na projekte mierne pozmeniť podľa okolností, mali by ste však mať počiatočnú predstavu. K návrhu vám dáme spätnú väzbu, pričom v niektorých prípadoch môže byť potrebné tému mierne alebo úplne zmeniť. Za načas odovzdaný vhodný návrh projektu získate 5% z celkovej známky. Návrh odporúčame pred odovzdaním konzultovať s vyučujúcimi.

Cez skúškové obdobie bude určený termín '''odovzdania projektu'''.  Podobne ako pri domácich úlohách odovzdávajte adresár s požadovanými súbormi:
* Vaše '''programy a súbory s dátami''' (veľmi veľké dátové súbory vynechajte) 
* '''Protokol''' podobne ako pri domácich úlohách 
** formát txt alebo pdf, stručné heslovité poznámky
** obsahuje zoznam súborov, podrobný postup pri analýze dát (spustené príkazy), ako aj použité zdroje (dáta, programy, dokumentácia a iná literatúra atď)
* '''Správu k projektu''' vo formáte pdf. Na rozdiel od menej formálneho protokolu by správu mal tvoriť súvislý text v odbornom štýle, podobne ako napr. záverečné práce. Môžete písať po slovensky alebo po anglicky, ale pokiaľ možno gramaticky správne. Správa by mala mať tieto časti:
** úvod, v ktorom vysvetlíte ciele projektu, prípadne potrebné poznatky zo skúmanej oblasti a aké dáta ste mali k dispozícii
** stručný popis metód, v ktorom neuvádzajte detailne jednotlivé kroky, skôr prehľad použitého prístupu a jeho zdôvodnenie
** výsledky analýzy (tabuľky, grafy a pod.) a popis týchto výsledkov, prípadne aké závery sa z nich dajú spraviť (nezabudnite vysvetliť, čo znamenajú údaje v tabuľkách, osi grafov a pod.). Okrem finálnych výsledkov analýzy uveďte aj čiastkové výsledky, ktorými ste sa snažili overovať, že pôvodné dáta a jednotlivé časti vášho postupu sa správajú rozumne. 
** diskusiu, v ktorej uvediete, ktoré časti projektu boli náročné a na aké problémy ste narazili, kde sa vám naopak podarilo nájsť spôsob, ako problém vyriešiť jednoducho, ktoré časti projektu by ste spätne odporúčali robiť iným než vašim postupom, čo ste sa na projekte naučili a podobne

Projekty môžete robiť aj vo '''dvojici''', vtedy však vyžadujeme rozsiahlejší projekt a každý člen by mal byť primárne zodpovedný za určitú časť projektu, čo uveďte aj v správe. Dvojice odovzdávajú jednu správu, ale po odovzdaní projektu majú stretnutie s vyučujúcimi individuálne.

Ako nájsť tému projektu:
* Môžete spracovať nejaké dáta, ktoré potrebujete do bakalárskej alebo diplomovej práce, prípadne aj dáta, ktoré potrebujte na iný predmet (v tom prípade uveďte v správe, o aký predmet ide a takisto upovedomte aj druhého vyučujúceho, že ste použili spracovanie dát ako projekt pre tento predmet). Obzvlášť pre BIN študentov môže byť tento predmet vhodnou príležitosťou nájsť si tému bakalárskej práce a začať na nej pracovať.
* Môžete skúsiť zopakovať analýzu spravenú v nejakom vedeckom článku a overiť, že dostanete tie isté výsledky. Vhodné je tiež skúsiť analýzu aj mierne obmeniť (spustiť na iné dáta, zmeniť nejaké nastavenia, zostaviť aj iný typ grafu a pod.)
* Môžete skúsiť nájsť niekoho, kto má dáta, ktoré by potreboval spracovať, ale nevie ako na to (môže ísť o biológov, vedcov z iných oblastí, ale aj neziskové organizácie a pod.) V prípade, že takýmto spôsobom kontaktujete tretie osoby, bolo by vhodné pracovať na projekte obzvlášť zodpovedne, aby ste nerobili zlé meno našej fakulte.
* V projekte môžete porovnávať niekoľko programov na tú istú úlohu z hľadiska ich rýchlosti či presnosti výsledkov. Obsahom projektu bude príprava dát, na ktorých budete programy bežať, samotné spúšťanie (vhodne zoskriptované) ako aj vyhodnotenie výsledkov.
* A samozrejme môžete niekde na internete vyhrabať zaujímavé dáta a snažiť sa z nich niečo vydolovať.

==Opisovanie==

* Máte povolené sa so spolužiakmi a ďalšími osobami rozprávať o domácich úlohách resp. projektoch a stratégiách na ich riešenie. Kód, získané výsledky aj text, ktorý odovzdáte, musí však byť vaša samostatná práca. Je zakázané ukazovať svoj kód alebo texty spolužiakom. 

* Pri riešení domácej úlohy a projektu očakávame, že budete využívať internetové zdroje, najmä rôzne manuály a diskusné fóra k preberaným technológiám. Nesnažte sa však nájsť hotové riešenia zadaných úloh. Všetky použité zdroje uveďte v domácich úlohách a projektoch. 

* Ak nájdeme prípady opisovania alebo nepovolených pomôcok, všetci zúčastnení študenti získajú za príslušnú domácu úlohu, projekt a pod. nula bodov (t.j. aj tí, ktorí dali spolužiakom odpísať) a prípad ďalej podstúpime na riešenie disciplinárnej komisii fakulty.

==Zverejňovanie==

Zadania a materiály k predmetu sú voľne prístupné na tejto stránke. Prosím vás ale, aby ste nezverejňovali ani inak nešírili vaše riešenia domácich úloh, ak nie je v zadaní povedané inak. Vaše projekty môžete zverejniť, pokiaľ to nie je v rozpore s vašou dohodou so zadávateľom projektu a poskytovateľom dát.
<!-- /NOTEX -->

=Lperl=
This lecture is a brief introduction to the Perl scripting language. More information can be found below (section [[#Sources of Perl-related information]]). We recommend revisiting necessary parts of this lecture while working on the exercises.

==Why Perl==
* From [https://en.wikipedia.org/wiki/Perl Wikipedia:] It has been nicknamed "the Swiss Army chainsaw of scripting languages" because of its flexibility and power, and possibly also because of its "ugliness".

Official slogans:
* There's more than one way to do it
* Easy things should be easy and hard things should be possible

Advantages
* Good capabilities for processing text files, regular expressions, running external programs etc.
* Closer to common programming languages than shell scripts
* Perl one-liners on the command line can replace many other tools such as sed and awk
* Many existing libraries

Disadvantages
* Quirky syntax
* It is easy to write very unreadable programs (Perl is sometimes joking called write-only language)
* Quite slow and uses a lot of memory. If possible, do no read entire input to memory, process line by line

We will use Perl 5, Perl 6 is quite a different language

==Hello world==
It is possible to run the code directly from a command line (more later):
<pre>
perl -e'print "Hello world\n"'
</pre>

This is equivalent to the following code stored in a file:
<pre>
#! /usr/bin/perl -w
use strict;
print "Hello world!\n";
</pre>

* The first line is a path to the interpreter
* Swith <tt>-w</tt> switches warnings on, e.g. if we manipulate with an undefined value (equivalent to <tt>use warnings;</tt>)
* The second line <tt>use strict</tt> will switch on a more strict syntax checks, e.g. all variables must be defined
* Use of <tt>-w</tt> and <tt>use strict</tt> is strongly recommended

Running the script
* Store the program in a file <tt>hello.pl</tt>
* Make it executable (<tt>chmod a+x hello.pl</tt>)
* Run it with command <tt>./hello.pl</tt>
* It is also possible to run as <tt>perl hello.pl</tt> (e.g. if we don't have the path to the interpreter in the file or the executable bit is not set)

==The first input file for today: sequence repeats==
* In genomes some sequences occur in many copies (often not exactly equal, only similar)
* We have downloaded a table containing such sequence repeats on chromosome 2L of the fruitfly ''Drosophila melanogaster''
* It was done as follows: on webpage [http://genome.ucsc.edu/ http://genome.ucsc.edu/] we select drosophila genome, then in main menu select  Tools, Table browser, select group: variation and repeats, track: ReapatMasker, region: position chr2L, output format: all fields from the selected table a output file: repeats.txt
* Each line of the file contains data about one repeat in the selected chromosome. The first line contains column names. Columns are tab-separated.
* Here are the first two lines, each line split into three lines for better readability
<pre>
#bin    swScore milliDiv        milliDel        milliIns
  genoName        genoStart       genoEnd genoLeft        strand
  repName repClass        repFamily       repStart        repEnd  repLeft id
585     778     167     7       20
  chr2L   1       154     -23513558  +
  HETRP_DM        Satellite       Satellite       1519    1669    -203    1
</pre>
* The file can be found at our server under filename <tt>/tasks/perl/repeats.txt</tt> (17185 lines)
* A small randomly selected subset of the table rows is in file <tt>/tasks/perl/repeats-small.txt</tt> (159 lines)

==A sample Perl program==
For each type of repeat (column 11 of the file when counting from 0) we want to compute the number of repeats of this type
<pre>
#!/usr/bin/perl -w
use strict;

#associative array (hash), with repeat type as key
my %count;  

while(my $line = <STDIN>) {  # read every line on input
    chomp $line;    # delete end of line, if any

    if($line =~ /^#/) {  # skip commented lines
       next;       # similar to "continue" in C, move to next iteration
    }

    # split the input line to columns on every tab, store them in an array
    my @columns = split "\t", $line;  

    # check input - should have at least 17 columns
    die "Bad input '$line'" unless @columns >= 17;

    my $type = $columns[11];

    # increase counter for this type
    $count{$type}++;
}

# write out results, types sorted alphabetically
foreach my $type (sort keys %count) {
    print $type, " ", $count{$type}, "\n";
}
</pre>

This program does the same thing as the following one-liner (more on one-liners in two weeks)
<pre>
perl -F'"\t"' -lane 'next if /^#/; die unless @F>=17; $count{$F[11]}++;
  END { foreach (sort keys %count) { print "$_ $count{$_}" }}' filename
</pre>


==The second input file for today: DNA sequencing reads (fastq)==

* DNA sequencing machines can read only short pieces of DNA called reads
* Reads are usually stored in [https://en.wikipedia.org/wiki/FASTQ_format FASTQ format]
* Files can be very large (gigabytes or more), but we will use only a small sample from bacteria ''Staphylococcus aureus'' (data from the [http://gage.cbcb.umd.edu/data/Staphylococcus_aureus/Data.original/ GAGE website])
* Each read is stored in 4 lines:
** line 1: ID of the read and other description, line starts with @
** line 2: DNA sequence, A,C,G,T are bases (nucleotides) of DNA, N means unknown base
** line 3: +
** line 4: quality string, which is the string of the same length as DNA in line 2. Each character represents quality of one base in DNA. If ''p'' is the probability that this base is wrong, the quality string will contain character with ASCII value 33+(-10 log ''p''), where log is the decimal logarithm. Higher ASCII means base of higher quality. Character <tt>!</tt> (ASCII 33) means probability 1 of error, character <tt>$</tt> (ASCII 36) means 50% error, character <tt>+</tt> (ASCII 43) is 10% error, character <tt>5</tt> (ASCII 53) is 1% error.
* Our file has all reads of equal length (this is not always the case)
* Technically, a single read and its quality can be split into multiple lines, but this is rarely done, and we will assume that each read takes 4 lines as described above

The first 4 reads from file <tt>/tasks/perl/reads-small.fastq</tt> (trimmed to 50 bases for better readability)
<pre>
@SRR022868.1845/1
AAATTTAGGAAAAGATGATTTAGCAACATTTAGCCTTAATGAAAGACCAG
+
IICIIIIIIIIIID%IIII8>I8III1II,II)I+III*II<II,E;-HI
@SRR022868.1846/1
TAGCGTTGTAAAATAAATTTCTAGAATGGAAGTGATGATATTGAAATACA
+
4CIIIIIIII52I)IIIII0I16IIIII2IIII;IIAII&I6AI+*+&G5
</pre>

==Variables, types==

===Scalar variables===
* The names of scalar variables start with <tt>$</tt> 
* Scalar variables can hold undefined value (<tt>undef</tt>), string, number, reference etc.
* Perl converts automatically between strings and numbers
<pre>
perl -e'print((1 . "2")+1, "\n")'
13
perl -e'print(("a" . "2")+1, "\n")'
1
perl -we'print(("a" . "2")+1, "\n")'
Argument "a2" isn't numeric in addition (+) at -e line 1.
1
</pre>
* If we switch on strict parsing, each variable needs to be defined by <tt>my</tt>
** Several variables can be created and initialized as follows: <tt>my ($a,$b) = (0,1);</tt>
* Usual set of C-style [http://perldoc.perl.org/perlop.html operators], power is <tt>**</tt>, string concatenation <tt>.</tt>
* Numbers compared by <tt><, <=, ==, !=</tt> etc., strings by <tt>lt, le, eq, ne, gt, ge</tt>  
* Comparison operator <tt>$a cmp $b</tt> for strings, <tt>$a <=> $b</tt> for numbers: returns -1 if <tt>$a<$b</tt>, 0 if they are equal, +1 if <tt>$a>$b</tt>

===Arrays===
* Names start with <tt>@</tt>, e.g. <tt>@a</tt>
* Access to element 0 in array <tt>@a</tt>: <tt>$a[0]</tt>
** Starts with <tt>$</tt>, because the expression as a whole is a scalar value
* Length of array <tt>scalar(@a)</tt>. In scalar context, <tt>@a</tt> is the same thing.
** e.g. <tt>for(my $i=0; $i<@a; $i++) { ... }</tt> iterates over all elements
* If using non-existent indexes, they will be created, initialized to <tt>undef</tt> (<tt>++, +=</tt> treat <tt>undef</tt> as 0)
* Stack/vector using functions <tt>push</tt> and <tt>pop</tt>: <tt>push @a, (1,2,3); $x = pop @a;</tt>
* Analogicaly <tt>shift</tt> and <tt>unshift</tt> on the left end of the array (slower)
* Sorting
** <tt>@a = sort @a;</tt> (sorts alphabetically)
** <tt>@a = sort {$a <=> $b} @a;</tt> (sorts numerically)
** <tt>{ }</tt> can contain an arbitrary comparison function, <tt>$a</tt> and <tt>$b</tt> are the two compared elements
* Array concatenation <tt>@c = (@a,@b);</tt>
* Swap values of two variables: <tt>($x,$y) = ($y,$x);</tt>
* Command <tt>foreach</tt> iterates through values of an array (values can be changed during iteration):
<pre>
my @a = (1,2,3);
foreach my $val (@a) {  # iterate through all values
    $val++;             # increase each value in array by 1
}
# concatenate values to a string separated by spaces
print join(" ", @a), "\n"; 
# prints 2 3 4
</pre>

===Hash tables (associative array, dictionaries, maps)===
* Names start with <tt>%</tt>, e.g. <tt>%b</tt>
* Keys are strings, values are scalars
* Access element with key <tt>"X"</tt>: <tt>$b{"X"}</tt>
* Write out all elements of associative array <tt>%b</tt>
<pre>
foreach my $key (keys %b) {
    print $key, " ", $b{$key}, "\n";
}
</pre>
* Initialization with a constant: <tt>%b = ("key1" => "value1", "key2" => "value2");</tt>
* Test for existence of a key: <tt>if(exists $a{"X"}) {...}</tt>

===Multidimensional arrays, fun with pointers===
* Pointer to a variable (scalar, array, dictionary): <tt>\$a, \@a, \%a</tt>
* Pointer to an anonymous array: <tt>[1,2,3]</tt>, pointer to an anonymous hash: <tt>{"key1" => "value1"}</tt>
* Hash of lists is stored as hash of pointers to lists:
<pre>
my %a = ("fruits" => ["apple","banana","orange"],
         "vegetables" => ["tomato","carrot"]);
$x = $a{"fruits"}[1];
push @{$a{"fruits"}}, "kiwi";
my $aref = \%a;
$x = $aref->{"fruits"}[1];
</pre>
* Module <tt>Data::Dumper</tt> has function <tt>Dumper</tt>, which recursively prints complex data structures (good for debuging)

==Strings==
* Substring: <tt>[http://perldoc.perl.org/functions/substr.html substr]($string, $start, $length)</tt>
** Used also to access individual characters (use length 1)
** If we omit <tt>$length</tt>, extracts suffix until the end of the string, negative <tt>$start</tt> counts from the end of the string,...
** We can also replace a substring by something else: <tt>substr($str, 0, 1) = "aaa"</tt> (replaces the first character by "aaa")
* Length of a string: <tt>[http://perldoc.perl.org/functions/length.html length]($str)</tt>
* Splitting a string to parts: <tt>[http://perldoc.perl.org/functions/split.html split] reg_expression, $string, $max_number_of_parts</tt>
** If <tt>" "</tt> is used instead of regular expression, splits at any whitespace
* Connecting parts to a string <tt>[http://perldoc.perl.org/functions/join.html join]($separator, @strings)</tt>
* Other useful functions: <tt>[http://perldoc.perl.org/functions/chomp.html chomp]</tt> (removes the end of line), <tt>[http://perldoc.perl.org/functions/index.html index]</tt> (finds a substring), <tt>lc</tt>, <tt>uc</tt> (conversion to lower-case/upper-case), <tt>reverse</tt> (mirror image), <tt>sprintf</tt> (C-style formatting)

==Regular expressions==
* Regular expressions are powerful tool for working with strings, now featued in many languages
* Here only a few examples, more details can be found in [http://perldoc.perl.org/perlretut.html the official tutorial]
<pre>
$line =~ s/\s+$//;      # remove whitespace at the end of the line
$line =~ s/[0-9]+/X/g;  # replace each sequence of numbers with character X

# if the line starts with >,
# store the word following > (until the first whitespace)
# and store it in variable $name 
# (\S means non-whitespace),
# the string matching part of expression in (..) is stroed in $1
if($line =~ /^\>(\S+)/) { $name = $1; }
</pre>

==Conditionals, loops==
<pre>
if(expression) {  # () and {} cannot be omitted
   commands
} elsif(expression) {
   commands
} else {
   commands
}

command if expression;   # here () not necessary
command unless expression;
# good for checking inputs etc
die "negative value of x: $x" unless $x >= 0;

for(my $i=0; $i<100; $i++) {
   print $i, "\n";
}

foreach my $i (0..99) {
   print $i, "\n";
}

my $x = 1;
while(1) {
   $x *= 2;
   last if $x >= 100;
}
</pre>

Undefined value, number 0 and strings <tt>""</tt> and <tt>"0"</tt> evaluate as false, but we recommmend always explicitly using logical values in conditional expressions, e.g. <tt>if(defined $x)</tt>, <tt>if($x eq "")</tt>, <tt>if($x==0)</tt> etc.

==Input, output==
* Reading one line from standard input: 
<pre>$line = <STDIN></pre>
* If no more input data available, returns <tt>undef</tt>
* See also [http://perldoc.perl.org/perlop.html#I%2fO-Operators|manual on Perl I/O operators]
* The special idiom below reads all the lines from input until the end of input is reached: 
<pre>
while (my $line = <STDIN>) { ... }  
</pre>
* <tt>chomp $line</tt> removes <tt>"\n"</tt>, if any from the end of the string
* Output to stdout through <tt>[http://perldoc.perl.org/functions/print.html print]</tt> or <tt>[http://perldoc.perl.org/functions/printf.html printf]</tt> commands

==Sources of Perl-related information==
* Man pages (included in ubuntu package <tt>perl-doc</tt>), also available online at [http://perldoc.perl.org/ http://perldoc.perl.org/]
** <tt>man perlintro</tt> introduction to Perl
** <tt>man perlfunc</tt> list of standard functions in Perl
** <tt>perldoc -f split</tt> describes function split, similarly other functions
** <tt>perldoc -q sort</tt> shows answers to commonly asked questions (FAQ)
** <tt>man perlretut</tt> and <tt>man perlre</tt> regular expressions
** <tt>man perl</tt> list of other manual pages about Perl
* Various web tutorials e.g. [http://www.perl.com/pub/a/2000/10/begperl1.html this one]
* Books
** [http://www.perl.org/books/beginning-perl/ Simon Cozens: Beginning Perl] freely downloadable
** [http://oreilly.com/catalog/9780596000271/ Larry Wall et al: Programming Perl] classics, Camel book

==Further optional topics==
For illustration, we briefly cover other topics frequently used in Perl scripts (tthese are not needed to solve the exercises).
===Opening files===
<pre>
my $in;
open $in, "<", "path/file.txt" or die;  # open file for reading
while(my $line = <$in>) {
  # process line
}
close $in;

my $out;
open $out, ">", "path/file2.txt" or die; # open file for writing
print $out "Hello world\n";
close $out;
# if we want to append to a file use the following instead:
# open $out, ">>", "cesta/subor2.txt" or die;

# standard files
print STDERR "Hello world\n";
my $line = <STDIN>;
# files as arguments of a function
read_my_file($in);
read_my_file(\*STDIN);
</pre>

===Working with files and directories===
Module <tt>File::Temp<tt> llows to create temporary working directories or files with automatically generated names. These are automatically deleted when the program finishes.
<pre>
use File::Temp qw/tempdir/;
my $dir = tempdir("atoms_XXXXXXX", TMPDIR => 1, CLEANUP => 1 ); 
print STDERR "Creating temporary directory $dir\n";
open $out,">$dir/myfile.txt" or die;
</pre>

Copying files
<pre>
use File::Copy;
copy("file1","file2") or die "Copy failed: $!";
copy("Copy.pm",\*STDOUT);
move("/dev1/fileA","/dev2/fileB");
</pre>
Other functions for working with file system, e.g. <tt>chdir, mkdir, unlink, chmod,</tt> ...

Function <tt>glob</tt> finds files with wildcard characters similarly as on command line (see also <tt>opendir, readdir</tt>, and <tt>File::Find module</tt>)
<pre>
ls *.pl
perl -le'foreach my $f (glob("*.pl")) { print $f; }'
</pre>

Additional functions for working with file names, paths, etc. in modules <tt>File::Spec</tt> and <tt>File::Basename</tt>.

Testing for an existence of a file (more in [http://perldoc.perl.org/functions/-X.html perldoc -f -X])
<pre>
if(-r "file.txt") { ... }  # is file.txt readable?
if(-d "dir") {.... }       # is dir a directory?
</pre>

===Running external programs===
Using the system command
* It returns -1 if it cannot run command, otherwise returns the return code of the program
<pre>
my $ret = system("command arguments");
</pre>

Using the backtick operator with capturing standard output to a variable
* This does not tests the return code
<pre>
my $allfiles = `ls`;
</pre>

Using pipes (special form of open sends output to a different command,
or reds output of a different command as a file)
<pre>
open $in, "ls |";
while(my $line = <$in>) { ... }
</pre>

<pre>
open $out, "| wc"; 
print $out "1234\n"; 
close $out;'

      1       1       5
</pre>

===Command-line arguments===
<pre>
# module for processing options in a standardized way
use Getopt::Std;
# string with usage manual
my $USAGE = "$0 [options] length filename

Options:
-l           switch on lucky mode
-o filename  write output to filename
";

# all arguments to the command are stored in @ARGV array
# parse options and remove them from @ARGV
my %options;
getopts("lo:", \%options);
# now there should be exactly two arguments in @ARGV
die $USAGE unless @ARGV==2;
# process options
my ($length, $filenamefile) = @ARGV;
# values of options are in the %options array
if(exists $options{'l'}) { print "Lucky mode\n"; }
</pre>
For long option names, see module Getopt::Long

===Defining functions===

<pre>
sub function_name {
  # arguments are stored in @_ array
  my ($firstarg, $secondarg) = @_;
  # do something
  return ($result, $second_result);
}
</pre>
* Arrays and hashes are usually passed as references: <tt>function_name(\@array, \%hash);</tt>
* It is advantageous to pass very long string as references to prevent needless copying: <tt>function_name(\$sequence);</tt>
* References need to be dereferenced, e.g. <tt>substr($$sequence)</tt> or <tt>$array->[0]</tt>

===Bioperl===
A large library useful for bioinformatics. This snippet translates DNA sequence to a protein using the standard genetic code:
<pre>
use Bio::Tools::CodonTable;
sub translate
{
    my ($seq, $code) = @_;
    my $CodonTable = Bio::Tools::CodonTable->new( -id => $code);
    my $result = $CodonTable->translate($seq);

    return $result;
}
</pre>


==HWperl==
<!-- NOTEX -->
See [[#Lperl|the lecture]]
<!-- /NOTEX -->

===Files and setup===
We recommend creating a directory (folder) for this set of tasks:
<pre>
mkdir perl  # make directory
cd perl    # change to the new directory
</pre>
We have 4 input files for this task set. We recommend creating soft links to your working directory as follows:
<pre>
ln -s /tasks/perl/repeats-small.txt .  # small version of the repeat file
ln -s /tasks/perl/repeats.txt .        # full version of the repeat file
ln -s /tasks/perl/reads-small.fastq .  # smaller version of the read file
ln -s /tasks/perl/reads.fastq .        # bigger version of the read file
</pre>

<!-- NOTEX -->
We recommend writing your protocol starting from an outline provided in  <tt>/tasks/perl/protocol.txt</tt>. Make your own copy of the protopcol and open it in an editor, e.g. kate:
<pre>
cp -ip /tasks/perl/protocol.txt . # copy protocol
kate protocol.txt &                # open editor, run in the backgrund
</pre>

===Submitting===
* Directory /submit/perl/your_username will be created for you
* Copy required files to this directory, including the protocol named protocol.txt or protocol.pdf
* You can modify these files freely until deadline, but after the deadline of the homework, you will lose access rights to this directory
<!-- /NOTEX -->

===Task A (repeats)===

* Consider the program for counting repeat types in the [[#Lperl#A_sample_Perl_program|lecture 1]], save it to file <tt>repeat-stat.pl</tt>
** Open editor running in the background: <tt>kate repeat-stat.pl</tt>
** Copy and paste text to the editor, save it 
** Make the script executable: <tt>chmod a+x repeat-stat.pl<//tt>
* Extend the script to compute the average length of each type of repeat
** Each row of the input table contains the start and end coordinates of the repeat in columns 7 and 6. The length is simply the difference of these two values.
* Output a table with three columns: type of repeat, the number of occurrences, the average length of the repeat. 
** Use [http://perldoc.perl.org/functions/printf.html printf] to print these three items right-justified in columns of sufficient width, print the average length to 1 decimal place.
* If you run your script on the small file, the output should look something like this (exact column widths may differ):
<pre>
./repeat-stat.pl < repeats-small.txt
                 DNA         5     377.4
                LINE         4     410.2
                 LTR        13     355.4
      Low_complexity        22      47.2
                  RC         8     236.2
       Simple_repeat       106      39.0
</pre>
* Run your script also on the large file: <tt>./repeat-stat.pl < repeats.txt</tt>
<!-- NOTEX -->
** Include the output in your '''protocol'''
<!-- /NOTEX -->
* Find out on [https://en.wikipedia.org/wiki/Retrotransposon Wikipedia], what acronyms LINE and LTR stand for. Do their names correspond to their lengths? 
<!-- NOTEX -->
** (Write a short answer in the '''protocol'''.) 
* '''Submit''' only your script, <tt>repeat-stat.pl</tt>
<!-- /NOTEX -->

===Task B (FASTQ to FASTA)===

* Write a script which reformats FASTQ file to FASTA format, call it <tt>fastq2fasta.pl</tt>
** [[#Lperl#The_second_input_file_for_today:_DNA_sequencing_reads_.28fastq.29|FASTQ file]] should be on standard input, FASTA file written to standard output
* [https://en.wikipedia.org/wiki/FASTA_format FASTA format] is a typical format for storing DNA and protein sequences. 
** Each sequence consists of several lines of the file. The first line starts with ">" followed by identifier of the sequence and optionally some further description separated by whitespace
** The sequence itself is on the second line, long sequences are split into multiple lines
* In our case, the name of the sequence will be the ID of the read with @ replaced by > and / replaced by underscore (<tt>_</tt>)
** you can try to use [http://perldoc.perl.org/perlop.html#Quote-Like-Operators tr or s operators] (see also [[#Lperl#Regular_expressions|lecture]])
* For example, the first two reads of the file <tt>reads.fastq</tt> are as follows (only the first 50 columns shown)
<pre>
@SRR022868.1845/1
AAATTTAGGAAAAGATGATTTAGCAACATTTAGCCTTAATGAAAGACCAG...
+
IICIIIIIIIIIID%IIII8>I8III1II,II)I+III*II<II,E;-HI...
@SRR022868.1846/1
TAGCGTTGTAAAATAAATTTCTAGAATGGAAGTGATGATATTGAAATACA...
+
4CIIIIIIII52I)IIIII0I16IIIII2IIII;IIAII&I6AI+*+&G5...
</pre>
* These should be reformatted as follows (again only first 50 columns shown, but you include entire reads):
<pre>
>SRR022868.1845_1
AAATTTAGGAAAAGATGATTTAGCAACATTTAGCCTTAATGAAAGACCAGA...
>SRR022868.1846_1
TAGCGTTGTAAAATAAATTTCTAGAATGGAAGTGATGATATTGAAATACAC...
</pre>
* Run your script on the small read file <tt>./fastq2fasta.pl < reads-small.fastq > reads-small.fasta</tt>
<!-- NOTEX -->
* '''Submit''' files <tt>fastq2fasta.pl</tt> and <tt>reads-small.fasta</tt> 
<!-- /NOTEX -->

===Task C (FASTQ quality)===

Write a script <tt>fastq-quality.pl</tt> which for each position in a read computes the average quality
* Standard input has fastq file with multiple reads, possibly of different lengths
* As quality we will use ASCII values of characters in the quality string with value 33 subtracted, so the quality is -10 log p
** ASCII value can be computed by function [http://perldoc.perl.org/functions/ord.html ord]
* Positions in reads will be numbered from 0
* Since reads can differ in length, some positions are used in more reads, some in fewer
* For each position from 0 up to the highest position used in some read, print three numbers separated by tabs "\t": the position index, the number of times this position was used in reads, the average quality at that position with 1 decimal place (you can again use <tt>printf</tt>)
* The last two lines when you run <tt>./fastq-quality.pl < reads-small.fastq</tt> should be
<pre>
99      86      5.5
100     86      8.6
</pre>
Run the following command, which runs your script on the larger file and selects every 10th position. 
<pre>
./fastq-quality.pl < reads.fastq | perl -lane 'print if $F[0]%10==0'
</pre>
* What trends (if any) do you see in quality values with increasing position?
<!-- NOTEX -->
* '''Submit''' only <tt>fastq-quality.pl</tt>
* In your '''protocol''', include the output of the command and the answer to the question above.
<!-- /NOTEX -->

===Task D (FASTQ trim)===

Write script <tt>fastq-trim.pl</tt> that trims low quality bases from the end of each read and filters out short reads
* This script should read a fastq file from standard input and write trimmed fastq file to standard output
* It should also accept two command-line arguments: character ''Q'' and integer ''L''
** We have not covered processing command line arguments, but you can use the code snippet below
* ''Q'' is the minimum acceptable quality (characters from quality string with ASCII value >= ASCII value of ''Q'' are ok)
* ''L'' is the minimum acceptable length of a read
* First find the last base in a read which has quality at least Q (if any). All bases after this base will be removed from both the sequence and quality string
* If the resulting read has fewer than L bases, it is omitted from the output

You can check your program by the following tests:
* If you run the following two commands, you should get file <tt>tmp</tt> identical with input and thus output of the <tt>diff</tt> command should be empty
<pre>
./fastq-trim.pl '!' 101 < reads-small.fastq > tmp  # trim at quality ASCII >=33 and length >=101
diff reads-small.fastq tmp                         # output should be empty (no differences)
</pre>

* If you run the following two commands, you should see differences in 4 reads, 2 bases trimmed from each
<pre>
./fastq-trim.pl '"' 1 < reads-small.fastq > tmp   # trim at quality ASCII >=34 and length >=1
diff reads-small.fastq tmp                        # output should be differences in 4 reads
</pre>

* If you run the following commands, you should get empty output (no reads meet the criteria):
<pre>
./fastq-trim.pl d 1 < reads-small.fastq           # quality ASCII >=100, length >= 1
./fastq-trim.pl '!' 102 < reads-small.fastq       # quality ASCII >=33 and length >=102
</pre>

<!-- NOTEX -->
Further runs and submitting
* <tt>./fastq-trim.pl '(' 95 < reads-small.fastq > reads-small-filtered.fastq  # quality ASCII >= 40</tt>  
* '''Submit''' files <tt>fastq-trim.pl</tt> and <tt>reads-small-filtered.fastq</tt>
<!-- /NOTEX -->
* If you have done task C, run quality statistics on the trimmed version of the bigger file using command below. Comment on the differences between statistics on the whole file in part C and D. Are they as you expected?
<pre>
# "2" means quality ASCII >= 50
./fastq-trim.pl 2 50 < reads.fastq | ./fastq-quality.pl | perl -lane 'print if $F[0]%10==0'
</pre>
<!-- NOTEX -->
* In your '''protocol''', include the result of the command and your discussion of its results.
<!-- /NOTEX -->

'''Note''': in this task set, you have created tools which can be combined, e.g. you can first trim FASTQ and then convert it to FASTA (no need to submit these files)

'''Parsing command-line arguments''' in this task (they will be stored in variables $Q and $L):
<pre>
#!/usr/bin/perl -w
use strict;

my $USAGE = "
Usage:
$0 Q L < input.fastq > output.fastq

Trim from the end of each read bases with ASCII quality value less
than the given threshold Q. If the length of the read after trimming
is less than L, the read will be omitted from output.

L is a non-negative integer, Q is a character
";

# check that we have exactly 2 command-line arguments
die $USAGE unless @ARGV==2;
# copy command-line arguments to variables Q and L
my ($Q, $L) = @ARGV;
# check that $Q is one character and $L looks like a non-negative integer
die $USAGE unless length($Q)==1 && $L=~/^[0-9]+$/;
</pre>

=Lbash=
<!-- NOTEX -->
[[#HWbash]]
<!-- /NOTEX -->

This lecture introduces command-line tools and Perl one-liners. 
* We will do simple transformations of text files using command-line tools without writing any scripts or longer programs. 

When working on the exercises, record all the commands used
* We strongly recommend making a log of commands for data processing also outside of this course
* If you have a log of executed commands, you can easily execute them again by copy and paste
* For this reason any comments are best preceded in the log by <tt>#</tt>
* If you use some sequence of commands often, you can turn it into a script

==Efficient use of the Bash command line==

Some tips for bash shell:
* use ''tab'' key to complete command names, path names etc 
** tab completion [https://www.debian-administration.org/article/316/An_introduction_to_bash_completion_part_1 can be customized]
* use ''up'' and ''down'' keys to walk through the history of recently executed commands, then edit and execute the chosen command
* press ''ctrl-r'' to search in the history of executed commands
* at the end of session, history stored in <tt>~/.bash_history</tt>
* command <tt>history -a</tt> appends history to this file right now
** you can then look into the file and copy appropriate commands to your log
* various other history tricks, e.g. special variables [http://samrowe.com/wordpress/advancing-in-the-bash-shell/]
* <tt>cd -</tt> goes to previously visited directory (also see <tt>pushd</tt> and <tt>popd</tt>)
* <tt>ls -lt | head</tt> shows 10 most recent files, useful for seeing what you have done last in a directory

Instead of bash, you can use more advanced command-line environments, e.g. [http://ipython.org/notebook.html iPhyton notebook]

==Redirecting and pipes==

<pre>
# redirect standard output to file
command > file

# append to file
command >> file

# redirect standard error
command 2>file

# redirect file to standard input
command < file

# do not forget to quote > in other uses, e.g. when searching for string ">" in a file sequences.fasta
grep '>' sequences.fasta
# (without quotes rewrites sequences.fasta)
# other special characters, such as ;, &, |, # etc
# should be quoted in '' as well

# send stdout of command1 to stdin of command2
command1 | command2

# backtick operator executes command, 
# removes trailing \n from stdout, substitutes to command line
# the following commands do the same thing:
head -n 2 file
head -n `echo 2` file

# redirect a string in ' ' to stdin of command head
head -n 2 <<< 'line 1
line 2
line 3'

# in some commands, file argument can be taken from stdin
# if denoted as - or stdin or /dev/stdin
# the following compares uncompressed version of file1 with file2
zcat file1.gz | diff - file2
</pre>

Make piped commands fail properly:
<pre>
set -o pipefail
</pre>
If set, the return value of a pipeline is the value of the last (rightmost) command to exit with a non-zero status, or zero if all commands in the pipeline exit successfully. This option is disabled by default, pipe then returns exit status of the rightmost command.

==Text file manipulation==
===Commands echo and cat (creating and printing files)===
<pre>
# print text Hello and end of line to stdout
echo "Hello" 
# interpret backslash combinations \n, \t etc:
echo -e "first line\nsecond\tline"
# concatenate several files to stdout
cat file1 file2
</pre>

===Commands head and tail (looking at start and end of files)===
<pre>
# print 10 first lines of file (or stdin)
head file
some_command | head 
# print the first 2 lines
head -n 2 file
# print the last 5 lines
tail -n 5 file
# print starting from line 100 (line numbering starts at 1)
tail -n +100 file
# print lines 81..100
head -n 100 file | tail -n 20 
</pre>
Documentation: [http://www.gnu.org/software/coreutils/manual/html_node/head-invocation.html head], [http://www.gnu.org/software/coreutils/manual/html_node/tail-invocation.html tail]

===Commands wc, ls -lh, od (exploring file statistics and details)===
<pre>
# prints three numbers:
# the number of lines (-l), number of words (-w), number of bytes (-c)
wc file

# prints the size of file in human-readable units (K,M,G,T)
ls -lh file

# od -a prints file or stdout with named characters 
#   allows checking whitespace and special characters
echo "hello world!" | od -a
# prints:
# 0000000   h   e   l   l   o  sp   w   o   r   l   d   !  nl
# 0000015
</pre>
Documentation: [http://www.gnu.org/software/coreutils/manual/html_node/wc-invocation.html wc], [http://www.gnu.org/software/coreutils/manual/html_node/ls-invocation.html ls], [http://www.gnu.org/software/coreutils/manual/html_node/od-invocation.html od]

===Command grep (getting lines matching a regular expression)===
<pre>
# get all lines containing string chromosome
grep chromosome file
# -i ignores case (upper case and lowercase letters are the same)
grep -i chromosome file
# -c counts the number of matching lines in each file
grep -c '^[12][0-9]' file1 file2

# other options (there is more, see the manual):
# -v print/count not matching lines (inVert)
# -n show also line numbers
# -B 2 -A 1 print 2 lines before each match and 1 line after match
# -E extended regular expressions (allows e.g. |)
# -F no regular expressions, set of fixed strings
# -f patterns in a file 
#    (good for selecting e.g. only lines matching one of "good" ids)
</pre>
Documentation: [http://www.gnu.org/software/grep/manual/grep.html grep]

===Commands sort, uniq===
<pre>
# sort lines of a file alphabetically
sort file

# some useful options of sort:
# -g numeric sort
# -k which column(s) to use as key
# -r reverse (from largest values)
# -s stable
# -t fields separator

# sorting first by column 2 numerically (-k2,2g),
# in case of ties use column 1 (-k1,1)
sort -k2,2g -k1,1 file 

# uniq outputs one line from each group of consecutive identical lines
# uniq -c adds the size of each group as the first column
# the following finds all unique lines
# and sorts them by frequency from the most frequent
sort file | uniq -c | sort -gr
</pre>
Documentation: [http://www.gnu.org/software/coreutils/manual/html_node/sort-invocation.html sort], [http://www.gnu.org/software/coreutils/manual/html_node/uniq-invocation.html uniq]

===Commands diff, comm (comparing files)===

Command <tt>[http://www.gnu.org/software/coreutils/manual/html_node/diff-invocation.html diff]</tt> compares two files. It is good for manual checking of differences. Useful options:
* <tt>-b</tt> (ignore whitespace differences) 
* <tt>-r</tt> for comparing whole directories
* <tt>-q</tt> for fast checking for identity
* <tt>-y</tt> show differences side-by-side

Command <tt>[http://www.gnu.org/software/coreutils/manual/html_node/comm-invocation.html comm]</tt> compares two sorted files. It is good for finding set intersections and differences. It writes three columns: 
* lines occurring only in the first file
* lines occurring only in the second file
* lines occurring in both files
Some columns can be suppressed with options <tt>-1, -2, -3</tt>


===Commands cut, paste, join (working with columns)===
* Command <tt>[http://www.gnu.org/software/coreutils/manual/html_node/cut-invocation.html cut]</tt> selects only some columns from file (perl/awk more flexible)
* Command <tt>[http://www.gnu.org/software/coreutils/manual/html_node/paste-invocation.html paste]</tt> puts two or more files side by side, separated by tabs or other characters
* Command <tt>[http://www.gnu.org/software/coreutils/manual/html_node/join-invocation.html join]</tt> is a powerful tool for making joins and left-joins as in databases on specified columns in two files

===Commands split, csplit (splitting files to parts)===
* Command <tt>[http://www.gnu.org/software/coreutils/manual/html_node/split-invocation.html split]</tt> splits into fixed-size pieces (size in lines, bytes etc.)
* Command <tt>[http://www.gnu.org/software/coreutils/manual/html_node/csplit-invocation.html csplit]</tt> splits at occurrence of a pattern. For example, splitting a FASTA file into individual sequences:
<pre>
csplit sequences.fa '/^>/' '{*}'
</pre>

==Programs sed and awk==
Both <tt>sed</tt> and <tt>awk</tt> process text files line by line, allowing to do various transformations
* <tt>awk</tt> newer, more advanced
* several examples below
* More info on [https://en.wikipedia.org/wiki/AWK awk], [https://en.wikipedia.org/wiki/Sed sed] on Wikipedia
<pre>
# replace text "Chr1" by "Chromosome 1"
sed 's/Chr1/Chromosome 1/'
# prints the first two lines, then quits (like head -n 2)
sed 2q  

# print the first and second column from a file
awk '{print $1, $2}' 

# print the line if the difference between the first and second column > 10
awk '{ if ($2-$1>10) print }'  

# print lines matching pattern
awk '/pattern/ { print }' 

# count the lines (like wc -l)
awk 'END { print NR }'
</pre>

==Perl one-liners==
Instead of sed and awk, we will cover Perl one-liners
* more examples on various websites ([http://www.math.harvard.edu/computing/perl/oneliners.txt example 1], [https://blogs.oracle.com/ksplice/entry/the_top_10_tricks_of example 2])
* documentation for  [http://perldoc.perl.org/perlrun.html Perl switches]
<pre>
# -e executes commands
perl -e'print 2+3,"\n"'
perl -e'$x = 2+3; print $x, "\n"';

# -n wraps commands in a loop reading lines from stdin
# or files listed as arguments
# the following is roughly the same as cat:
perl -ne'print'
# how to use:
perl -ne'print' < input > output
perl -ne'print' input1 input2 > output
# lines are stored in a special variable $_
# this variable is default argument of many functions, 
# including print, so print is the same as print $_

# simple grep-like commands:
perl -ne 'print if /pattern/'
# simple regular expression modifications
perl -ne 's/Chr(\d+)/Chromosome $1/; print'
# // and s/// are applied by default to $_

# -l removes end of line from each input line and adds "\n" after each print
# the following adds * at the end of each line
perl -lne'print $_, "*"' 

# -a splits line into words separated by whitespace and stores them in array @F
# the next example prints difference in the numbers stored
# in the second and first column
# (e.g. interval size if each line coordinates of one interval)
perl -lane'print $F[1]-$F[0]'

# -F allows to set separator used for splitting (regular expression)
# the next example splits at tabs
perl -F '"\t"' -lane'print $F[1]-$F[0]'

# END { commands } is run at the very end, after we finish reading input
# the following example computes the sum of interval lengths
perl -lane'$sum += $F[1]-$F[0]; END { print $sum; }'
# similarly BEGIN { command } before we start
</pre>

Other interesting possibilites:
<pre>
# -i replaces each file with a new transformed version (DANGEROUS!)
# the next example removes empty lines from all .txt files
# in the current directory
perl -lne 'print if length($_)>0' -i *.txt
# the following example replaces sequence of whitespace by exactly one space 
# and removes leading and trailing spaces from lines in all .txt files
perl -lane 'print join(" ", @F)' -i *.txt

# variable $. contains the line number. $ARGV the name of file or - for stdin
# the following prints filename and line number in front of every line
perl -ne'printf "%s.%d: %s", $ARGV, $., $_' file1 file2

# moving files *.txt to have extension .tsv:
#   first print commands 
#   then execute by hand or replace print with system
#   mv -i asks if something is to be rewritten
ls *.txt | perl -lne '$s=$_; $s=~s/\.txt/.tsv/; print("mv -i $_ $s")'
ls *.txt | perl -lne '$s=$_; $s=~s/\.txt/.tsv/; system("mv -i $_ $s")'
</pre>

==HWbash==
<!-- NOTEX -->
[[#Lperl|Lecture on Perl]], [[#Lbash|Lecture on command-line tools]]
<!-- /NOTEX -->

* In this set of tasks, use command-line tools or one-liners in Perl, awk or sed. Do not write any scripts or programs. 
* Each task can be split into several stages and intermediate files written to disk, but you can also use pipelines to reduce the number of temporary files. 
* Your commands should work also for other input files with the same format (do not try to generalize them too much, but also do not use very specific properties of a particular input, such as the number of lines etc.)
<!-- NOTEX -->
* Include all relevant used commands in your protocol and add a short description of your approach.
* Submit the protocol and required output files.
* Outline of the protocol is in <tt>/tasks/bash/protocol.txt</tt>, submit to directory <tt>/submit/bash/yourname</tt>
<!-- /NOTEX -->

===Task A (passwords)===
* The file <tt>/tasks/bash/names.txt</tt> contains data about several people, one per line. 
* Each line consists of given name(s), surname and email separated by spaces. 
* Each person can have multiple given names (at least 1), but exactly one surname and one email. Email is always of the form <tt>username@uniba.sk</tt>.
* The task is to generate file <tt>passwords.csv</tt> which contains a randomly generated password for each of these users
** The output file has columns separated by commas ','
** The first column contains username extracted from email address, the second column surname, the third column all given names and the fourth column the randomly generated password
<!-- NOTEX -->
* '''Submit''' file <tt>passwords.csv</tt> with the result of your commands. 
<!-- /NOTEX -->

Example line from input:
<pre>
Pavol Országh Hviezdoslav hviezdoslav32@uniba.sk
</pre>

Example line from output (password will differ):
<pre>
hviezdoslav32,Hviezdoslav,Pavol Országh,3T3Pu3un
</pre>

Hints:
* Passwords can be generated using <tt>pwgen</tt> (e.g. <tt>pwgen -N 10 -1</tt> prints 10 passwords, one per line) 
* We also recommend using <tt>perl</tt>, <tt>wc</tt>, <tt>paste</tt> (check option <tt>-d</tt> in <tt>paste</tt>)
* In Perl, function <tt>[http://perldoc.perl.org/functions/pop.html pop]</tt> may be useful for manipulating <tt>@F</tt> and function <tt>[http://perldoc.perl.org/functions/join.html join]</tt> for connecting strings with a separator.

===Task B (yeast genome)===

'''The input file:'''
* <tt>/tasks/bash/saccharomyces_cerevisiae.gff</tt> contains annotation of the yeast genome 
** Downloaded from http://yeastgenome.org/ on 2016-03-09, in particular from [http://downloads.yeastgenome.org/curation/chromosomal_feature/saccharomyces_cerevisiae.gff]. 
** It was further processed to omit DNA sequences from the end of file. 
** The size of the file is 5.6M. 
* For easier work, link the file to your directory by <tt>ln -s /tasks/bash/saccharomyces_cerevisiae.gff yeast.gff</tt>
* The file is in [http://www.sequenceontology.org/gff3.shtml GFF3 format]
* The lines starting with <tt>#</tt> are comments, other lines contain tab-separated data about one interval of some chromosome in the yeast genome
* Meaning of the first 5 columns:
** column 0 chromosome name
** column 1 source (can be ignored)
** column 2 type of interval
** column 3 start of interval (1-based coordinates)
** column 4 end of interval (1-based coordinates)
* You can assume that these first 5 columns do not contain whitespace

'''Task:'''
* Print for each type of interval (column 2), how many times it occurs in the file. 
* Sort from the most common to the least common interval types.
* Hint: commands <tt>sort</tt> and <tt>uniq</tt> will be useful. Do not forget to skip comments, for example using <tt>grep -v '^#'</tt>
* The result should be a file  <tt>types.txt</tt> formatted as follows:
<pre>
   7058 CDS
   6600 mRNA
...
...
      1 telomerase_RNA_gene
      1 mating_type_region
      1 intein_encoding_region

</pre>
<!-- NOTEX -->
'''Submit''' the file <tt>types.txt</tt>
<!-- /NOTEX -->

===Task C (chromosomes)===
* Continue processing file from task B. 
* For each chromosome, the file contains a line which has in column 2 string <tt>chromosome</tt>, and the interval is the whole chromosome.
* To file <tt>chrosomes.txt</tt>, print a tab-separated list of chromosome names and sizes in the same order as in the input
* The last line of <tt>chromosomes.txt</tt> should list the total size of all chromosomes combined.
<!-- NOTEX -->
* '''Submit''' file <tt>chromosomes.txt</tt>
<!-- /NOTEX -->
* Hints: 
** The total size can be computed by a perl one-liner.
** Example from the lecture: compute the sum of interval sizes if each line of the file contains start and end of one interval: <tt>perl -lane'$sum += $F[1]-$F[0]; END { print $sum; }'</tt>
** Grepping for word chromosome does not check if this word is indeed in the second column
** Tab character is written in Perl as <tt>"\t"</tt>.
* Your output should start and end as follows:
<pre>
chrI    230218
chrII   813184
...
...
chrXVI  948066
chrmt   85779
total   12157105
</pre>

===Task D (blast)===
'''Overall goal:'''
* Proteins from several well-studied yeast species were downloaded from database http://www.uniprot.org/ on 2016-03-09. The file contains sequence of the protein as well as a short description of its biological function.
* We have also downloaded proteins from the yeast ''Yarrowia lipolytica''. We will pretend that nothing is known about the function of these proteins (as if they were produced by gene finding program in a newly sequenced genome).
* For each ''Y.lipolytica'' protein, we have found similar proteins from other yeasts
* Now we want to extract for each protein in ''Y.lipolytica'' its closest match among all known proteins and see what is its function. This will give a clue about the potential function of the ''Y.lipolytica'' protein.

'''Files:'''
* <tt>/tasks/bash/known.fa</tt> is a FASTA file containing sequences of known proteins from several species
* <tt>/tasks/bash/yarLip.fa</tt> is a FASTA file with proteins from ''Y.lipolytica''
* <tt>/tasks/bash/known.blast</tt> is the result of finding similar proteins in <tt>yarLip.fa</tt> versus <tt>known.fa</tt> by these commands (already done by us):
<pre>
formatdb -i known.fa
blastall -p blastp -d known.fa -i yarLip.fa -m 9 -e 1e-5 > known.blast
</pre>
* you can link these files to your directory as follows:
<pre>
ln -s /tasks/bash/known.fa .
ln -s /tasks/bash/yarLip.fa .
ln -s /tasks/bash/known.blast .
</pre>

'''Step 1:'''
* Get the first (strongest) match for each query from <tt>known.blast</tt>.
* This can be done by printing the lines that are not comments but follow a comment line starting with #. 
* In a Perl one-liner, you can create a state variable which will remember if the previous line was a comment and based on that you decide of you print the current line. 
* Instead of using Perl, you can play with grep. Option <tt>-A 1</tt> prints the matching lines as well as one line ofter each match
* Print only the first two columns separated by tab (name of query, name of target), sort the file by the second column.
* Store the result in file <tt>best.tsv</tt>. The file should start as follows:
<pre>
Q6CBS2  sp|B5BP46|YP52_SCHPO
Q6C8R4  sp|B5BP48|YP54_SCHPO
Q6CG80  sp|B5BP48|YP54_SCHPO
Q6CH56  sp|B5BP48|YP54_SCHPO
</pre>
<!-- NOTEX -->
* '''Submit''' file <tt>best.tsv</tt> with the result
<!-- /NOTEX -->

'''Step 2:'''
* Create file <tt>known.tsv</tt> which contains sequence names extracted from <tt>known.fa</tt> with leading <tt>></tt> removed
* This file should be sorted alphabetically.
* The file should start as follows (lines are trimmed below):
<pre>
sp|A0A023PXA5|YA19A_YEAST Putative uncharacterized protein YAL019W-A OS=Saccharomyces...
sp|A0A023PXB0|YA019_YEAST Putative uncharacterized protein YAR019W-A OS=Saccharomyces...
</pre>
* '''Submit''' file <tt>known.tsv</tt>

'''Step 3:'''
* Use command [http://www.gnu.org/software/coreutils/manual/html_node/join-invocation.html join] to join the files <tt>best.tsv</tt> and <tt>known.tsv</tt> so that each line of <tt>best.tsv</tt> is extended with the text describing the corresponding target in <tt>known.tsv</tt>
* Use option <tt>-1 2</tt> to use the second column of <tt>best.tsv</tt> as a key for joining
* The output of <tt>join</tt> may look as follows:
<pre>
sp|B5BP46|YP52_SCHPO Q6CBS2 Putative glutathione S-transferase C1183.02 OS=Schizosaccharomyces...
sp|B5BP48|YP54_SCHPO Q6C8R4 Putative alpha-ketoglutarate-dependent sulfonate dioxygenase OS=...
</pre>
* Further reformat the output so that the query name goes first (e.g. <tt>Q6CBS2</tt>), followed by target name (e.g. <tt>sp|B5BP46|YP52_SCHPO</tt>), followed by the rest of the text, but remove all text after <tt>OS=</tt>
* Sort by query name, store as <tt>best.txt</tt> 
* The output should start as follows:
<pre>
B5FVA8  tr|Q5A7D5|Q5A7D5_CANAL  Lysophospholipase
B5FVB0  sp|O74810|UBC1_SCHPO    Ubiquitin-conjugating enzyme E2 1
B5FVB1  sp|O13877|RPAB5_SCHPO   DNA-directed RNA polymerases I, II, and III subunit RPABC5
</pre>
<!-- NOTEX -->
* '''Submit''' file  <tt>best.txt</tt>
<!-- /NOTEX -->

'''Note:'''
* Not all ''Y.lipolytica'' proteins are necessarily included in your final output (some proteins do not have blast match).
** You can think how to find the list of such proteins, but this is not part of the task.
* Files <tt>best.txt</tt> and <tt>best.tsv</tt> should have the same number of lines.
<!-- TODO: some observations on the results -->

=Lmake=
==Job Scheduling==

* Some computing jobs take a lot of time: hours, days, weeks,...
* We do not want to keep a command-line window open the whole time; therefore we run such jobs in the background
* Simple commands to do it in Linux: 
** To run the program immediately, then switch the whole console to the background: [https://www.gnu.org/software/screen/manual/screen.html screen], [https://tmux.github.io/ tmux]
** To run the command when the computer becomes idle: [http://pubs.opengroup.org/onlinepubs/9699919799/utilities/batch.html batch]
* Now we will concentrate on '''[https://en.wikipedia.org/wiki/Oracle_Grid_Engine Sun Grid Engine]''', a complex software for managing many jobs from many users on a cluster consisting of multiple computers
* Basic workflow:
** Submit a job (command) to a queue
** The job waits in the queue until resources (memory, CPUs, etc.) become available on some computer
** The job runs on the computer
** Output of the job is stored in files
** User can monitor the status of the job (waiting, running)
* Complex possibilities for assigning priorities and deadlines to jobs, managing multiple queues etc.
* Ideally all computers in the cluster share the same environment and filesystem
<!-- NOTEX -->
* We have a simple training cluster for this exercise:
** You submit jobs to queue on vyuka
** They will run on computer cpu02 
** This cluster is only temporarily available until next Thursday
<!-- /NOTEX -->


===Submitting a job (qsub)===

Basic commad: <tt>qsub -b y -cwd 'command < input > output 2> error'</tt>
* quoting around command allows us to include special characters, such as <tt><, ></tt> etc. and not to apply it to <tt>qsub</tt> command itself
* <tt>-b y</tt> treats command as binary, usually preferable for both binary programs and scripts
* <tt>-cwd</tt> executes command in the current directory
* <tt>-N</tt> name allows to set name of the job
* <tt>-l resource=value</tt> requests some non-default resources
* for example, we can use <tt>-l threads=2</tt> to request 2 threads for parallel programs
* Grid engine will not check if you do not use more CPUs or memory than requested, be considerate (and perhaps occasionally watch your jobs by running top at the computer where they execute)
* qsub will create files for stdout and stderr, e.g. s2.o27 and s2.e27 for the job with name s2 and jobid 27

===Monitoring and deleting jobs (qstat, qdel)===
Command <tt>qstat</tt> displays jobs of the current user
* job 28 is running of  server cpu02 (status <t>r</tt>), job 29 is waiting in queue (status <tt>qw</tt>)
<pre>
job-ID  prior   name       user         state submit/start at     queue       
------------------------------------------------------------------------------
     28 0.50000 s3         bbrejova     r     03/15/2016 22:12:18 main.q@cpu02
     29 0.00000 s3         bbrejova     qw    03/15/2016 22:14:08             
</pre>

* Command <tt>qstat -u '*'</tt> displays jobs of all users
* Finished jobs disappear from the list
* Command <tt>qstat -F threads</tt> shows how many threads available
<pre>
queuename                      qtype resv/used/tot. load_avg arch          states
---------------------------------------------------------------------------------
main.q@cpu02.compbio.fmph.unib BIP   0/2/8          0.03     lx26-amd64
        hc:threads=0
     28 0.75000 s3         bbrejova     r     03/15/2016 22:12:18     1
     29 0.25000 s3         bbrejova     r     03/15/2016 22:14:18     1
</pre>

* Command <tt>qdel</tt> deletes a job (waiting or running)

===Interactive work on the cluster (qrsh), screen===
Command <tt>qrsh</tt> creates a job which is a normal interactive shell running on the cluster
* In this shell you can manually run commands
* When you close the shell, the job finishes
* therefore it is a good idea to run <tt>qrsh</tt> within <tt>screen</tt>
** run <tt>screen</tt> command, this creates a new shell 
** within this shell, run <tt>qrsh</tt>, then whatever commands
** by pressing <tt>Ctrl-a d</tt> you "detach" the screen, so that both shells (local and <tt>qrsh</tt>) continue running but you can close your local window
** later by running <tt>screen -r</tt> you get back to your shells

===Running many small jobs===
For example, we many need run some computation for each human gene (there are roughly 20,000 such genes). Here are some possibilties:
* Run a script which iterates through all jobs and runs them sequentially 
** Problems: Does not use parallelism, needs more programming to restart after some interruption
* Submit processing of each gene as a separate job to cluster (submitting done by a script/one-liner) 
** Jobs can run in parallel on many different computers
** Problem: Queue gets very long, hard to monitor progress, hard to resubmit only unfinished jobs after some failure.
* Array jobs in qsub (option <tt>-t</tt>): runs jobs numbered 1,2,3...; number of the current job is in an environment variable, used by the script to decide which gene to process
** Queue contains only running sub-jobs plus one line for the remaining part of the array job. 
** After failure, you can resubmit only unfinished portion of the interval (e.g. start from job 173).
* Next: using make in which you specify how to process each gene and submit a single make command to the queue
** Make can execute multiple tasks in parallel using several threads on the same computer (<tt>qsub</tt> array jobs can run tasks on multiple computers)
** It will automatically skip tasks which are already finished, so restart os easy

==Make==
[https://en.wikipedia.org/wiki/Make_(software) Make] is a system for automatically building programs (running compiler, linker etc)
* In particular, we will use [https://www.gnu.org/software/make/manual/ GNU make]
* Rules for compilation are written in a <tt>Makefile</tt> 
* Rather complex syntax with many features, we will only cover basics

===Rules===
* The main part of a <tt>Makefile</tt> are rules specifying how to generate target files from some source files (prerequisites). 
* For example the following rule generates file <tt>target.txt</tt> by concatenating files <tt>source1.txt</tt> and <tt>source2.txt</tt>:
<pre>
target.txt : source1.txt source2.txt
      cat source1.txt source2.txt > target.txt
</pre>
* The first line describes target and prerequisites, starts in the first column
* The following lines list commands to execute to create the target
* Each line with a command starts with a '''tab''' character

* If we have a directory with this rule in file called <tt>Makefile</tt> and files <tt>source1.txt</tt> and <tt>source2.txt</tt>, running <tt>make target.txt</tt> will run the <tt>cat</tt> command
* However, if <tt>target.txt</tt> already exists, the command will be run only if one of the prerequisites has more recent modification time than the target
* This allows to restart interrupted computations or rerun necessary parts after modification of some input files
* <tt>make</tt> automatically chains the rules as necessary: 
** if we run <tt>make target.txt</tt> and some prerequisite does not exist, <tt>make</tt> checks if it can be created by some other rule and runs that rule first
** In general it first finds all necessary steps and runs them in appropriate order so that each rules has its prerequisites ready
** Option <tt>make -n target</tt> will show which commands would be executed to build target (dry run) - good idea before running something potentially dangerous

===Pattern rules===

We can specify a general rule for files with a systematic naming scheme. For example, to create a <tt>.pdf</tt> file from a <tt>.tex</tt> file, we use the <tt>pdflatex</tt> command:
<pre>
%.pdf : %.tex
      pdflatex $^
</pre>
* In the first line, <tt>%</tt> denotes some variable part of the filename, which has to agree in the target and all prerequisites
* In commands, we can use several variables:
** Variable <tt>$^</tt> contains the names of the prerequisites (source)
** Variable <tt>$@</tt> contains the name of the target
** Variable <tt>$*</tt> contains the string matched by <tt>%</tt>

===Other useful tricks in Makefiles===

====Variables====
Store some reusable values in variables, then use them several times in the <tt>Makefile</tt>:
<pre>
MYPATH := /projects/trees/bin

target : source
       $(MYPATH)/script < $^ > $@
</pre>

====Wildcards, creating a list of targets from files in the directory====

The following <tt>Makefile</tt> automatically creates <tt>.png</tt> version of each <tt>.eps</tt> file simply by running <tt>make</tt>:
<pre>
EPS := $(wildcard *.eps)
EPSPNG := $(patsubst %.eps,%.png,$(EPS))

all:  $(EPSPNG)

clean:
        rm $(EPSPNG)

%.png : %.eps
        convert -density 250 $^ $@
</pre>
* variable <tt>EPS</tt> contains names of all files matching <tt>*.eps</tt>
* variable <tt>EPSPNG</tt> contains desirable names of <tt>.png</tt> files
** it is created by taking filenames in <tt>EPS</tt> and changing <tt>.eps</tt> to <tt>.png</tt>
* <tt>all</tt> is a "phony target" which is not really created
** its rule has no commands but all <tt>.png</tt> files are prerequisites, so are done first
** the first target in a <tt>Makefile</tt> (in this case <tt>all</tt>) is default when no other target is specified on the command-line
* <tt>clean</tt> is also a phony target for deleting generated <tt>.png</tt> files

====Useful special built-in target names====
Include these lines in your <tt>Makefile</tt> if desired
<pre>
.SECONDARY:
# prevents deletion of intermediate targets in chained rules

.DELETE_ON_ERROR:
# delete targets if a rule fails
</pre>

===Parallel make===
Running make with option <tt>-j 4</tt> will run up to 4 commands in parallel if their dependencies are already finished. Ths allows easy parallelization on a single computer.

==Alternatives to Makefiles==
* Bioinformaticians often uses "pipelines" - sequences of commands run one after another, e.g. by a script or <tt>make</tt>
* There are many tools developed for automating computational pipelines, see e.g. this review: [https://academic.oup.com/bib/article/doi/10.1093/bib/bbw020/2562749/A-review-of-bioinformatic-pipeline-frameworks Jeremy Leipzig; A review of bioinformatic pipeline frameworks. Brief Bioinform 2016.]
* For example [https://bitbucket.org/snakemake/snakemake/wiki/Home Snakemake]
** Snake workflows can contain shell commands or Python code
** Big advantage compared to <tt>make</tt>: pattern rules may contain multiple variable portions (in <tt>make</tt> only one <tt>%</tt> per filename)
** For example, assume we have several FASTA files and several profiles (HMMs) representing protein families and we want to run each profile on each FASTA file:
<pre>
rule HMMER:
     input: "{filename}.fasta", "{profile}.hmm"
     output: "{filename}_{profile}.hmmer"
     shell: "hmmsearch --domE 1e-5 --noali --domtblout {output} {input[1]} {input[0]}"
</pre>

==HWmake==

<!-- NOTEX -->
See also the [[#Lmake|lecture]]
<!-- /NOTEX -->

===Motivation: Building Phylogenetic Trees===
The task for today will be to build a phylogenetic tree of 9 mammalian species using protein sequences
* A '''phylogenetic tree''' is a tree showing evolutionary history of these species. Leaves are the present-day species, internal nodes are their common ancestors.
* The '''input''' contains sequences of all proteins from each species (we will use only a smaller subset)
* The process is typically split into severla stages shown below

====Identify ortholog groups====
''Orthologs'' are proteins from different species that "correspond" to each other. Ortholog are found based on sequence similarity and we can use a tool called [http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastDocs&DOC_TYPE=Download blast] to identify sequence similarities between pairs of proteins. The result of ortholog group identification will be a set of groups, each group having one sequence from each of the 9 species

====Align proteins on each group====
For each ortholog group, we need to align proteins in the group to identify corresponding parts of the proteins. This is done by a tool called <tt>muscle</tt>
 
Unaligned sequences (start of protein [https://www.uniprot.org/uniprot/O60568 O60568]):
<pre>
>human
MTSSGPGPRFLLLLPLLLPPAASASDRPRGRDPVNPEKLLVITVA...
>baboon
MTSSRPGLRLLLLLLLLPPAASASDRPRGRDPVNPEKLLVMTVA...
>dog
MASSGPGLRLLLGLLLLLPPPPATSASDRPRGGDPVNPEKLLVITVA...
>elephant
MASWGPGARLLLLLLLLLLPPPPATSASDRSRGSDRVNPERLLVITVA...
>guineapig
MAFGAWLLLLPLLLLPPPPGACASDQPRGSNPVNPEKLLVITVA...
>opossum
SDKLLVITAA...
>pig
AMASGPGLRLLLLPLLVLSPPPAASASDRPRGSDPVNPDKLLVITVA...
>rabbit
MGCDSRKPLLLLPLLPLALVLQPWSARGRASAEEPSSISPDKLLVITVA...
>rat
MAASVPEPRLLLLLLLLLPPLPPVTSASDRPRGANPVNPDKLLVITVA...
</pre>

Aligned sequences:
<pre>
rabbit       MGCDSRKPLL LLPLLPLALV LQPW-SARGR ASAEEPSSIS PDKLLVITVA ...
guineapig    MAFGA----W LLLLPLLLLP PPPGACASDQ PRGSNP--VN PEKLLVITVA ...
opossum      ---------- ---------- ---------- ---------- SDKLLVITAA ...
rat          MAASVPEPRL LLLLLLLLPP LPPVTSASDR PRGANP--VN PDKLLVITVA ...
elephant     MASWGPGARL LLLLLLLLLP PPPATSASDR SRGSDR--VN PERLLVITVA ...
human        MTSSGPGPRF LLLLPLLL-- -PPAASASDR PRGRDP--VN PEKLLVITVA ...
baboon       MTSSRPGLRL LLLLLLL--- -PPAASASDR PRGRDP--VN PEKLLVMTVA ...
dog          MASSGPGLRL LLGLLLLL-P PPPATSASDR PRGGDP--VN PEKLLVITVA ...
pig          AMASGPGLR- LLLLPLLVLS PPPAASASDR PRGSDP--VN PDKLLVITVA ...
</pre>

====Build phylogenetic tree for each grup====

For each alignment, we build a phylogenetic tree for this group. We will use a program called phyml.

Example of a phylogenetic tree in newick format:
<pre>
 ((opossum:0.09636245,rabbit:0.85794020):0.05219782,
(rat:0.07263127,elephant:0.03306863):0.01043531,
(dog:0.01700528,(pig:0.02891345,
(guineapig:0.14451043,
(human:0.01169266,baboon:0.00827402):0.02619598
):0.00816185):0.00631423):0.00800806);
</pre>

[[Image:Tree.png|center|thumb|200px|Tree for gene O60568 (note: this particular tree does not agree well with real evolutionary history)]] 

====Build a consensus tree====

The result of the previous step will be several trees, one for every group. Ideally, all trees would be identical, showing the real evolutionary history of the 9 species. But it is not easy to infer the real tree from sequence data, so the trees from different groups might differ. Therefore, in the last step, we will build a consensus tree. This can be done by using a tool called Phylip. The output is a single consensus tree.

<!-- NOTEX -->
===Files and submitting===
<!-- /NOTEX -->
<!-- TEX
===Files===
/TEX -->

Our goal for today is to build a pipeline that automates the whole task using make and execute it remotely using qsub. Most of the work is already done, only small modifications are necessary.

<!-- NOTEX -->
* Submit by copying requested files to <tt>/submit/make/username/</tt>
* Do not forget to submit protocol, outline of the protocol is in <tt>/tasks/make/protocol.txt</tt>
<!-- /NOTEX -->

Start by copying directory <tt>/tasks/make</tt> to your user directory
<pre>
cp -ipr /tasks/make .
cd make
</pre>

The directory contains three subdirectories:
* <tt>large</tt>: a larger sample of proteins for task A
* <tt>tiny</tt>: a very small set of proteins for task B
* <tt>small</tt>: a slightly larger set of proteins for task C

===Task A (long job)===

* In this task, you will run a long alignment job (more than two hours)
* Use directory <tt>large</tt> with files:
** <tt>ref.fa</tt>: selected human proteins
** <tt>other.fa</tt>: selected proteins from 8 other mammalian species
** <tt>Makefile</tt>: runs blast on <tt>ref.fa</tt> vs <tt>other.fa</tt> (also formats database <tt>other.fa</tt> before that)
* run <tt>make -n</tt> to see what commands will be done (you should see <tt>makeblastdb</tt>, <tt>blastp</tt>, and <tt>echo</tt> for timing) 
<!-- NOTEX -->
** copy the output to the '''protocol'''
<!-- /NOTEX -->
* run <tt>qsub</tt> with appropriate options to run <tt>make</tt> (at least <tt>-cwd -b y</tt>)
<!-- NOTEX -->
* then run <tt>qstat > queue.txt </tt>
** '''Submit''' file <tt>queue.txt</tt> showing your job waiting or running 
<!-- /NOTEX -->
<!-- TEX
* Use <tt>qsub</tt> to check the status of your job
/TEX -->
* When your job finishes, check the following files:
** the output file <tt>ref.blast</tt>
** standard output from the <tt>qsub</tt> job, which is stored in a file named e.g. <tt>make.oX</tt> where <tt>X</tt> is the number of your job. The output shows the time when your job started and finished (this information was written by commands <tt>echo</tt> in the <tt>Makefile</tt>)
<!-- NOTEX -->
* '''Submit''' the last 100 lines from <tt>ref.blast</tt> under the name </tt>ref-end.blast<tt> (use tool <tt>tail -n 100</tt>) and the file <tt>make.oX</tt> mentioned above 
<!-- /NOTEX -->

===Task B (finishing Makefile)===

* In this task, you will finish a <tt>Makefile</tt> for splitting blast results into ortholog groups and building phylogenetic trees for each group
** This <tt>Makefile</tt> works with much smaller files and so you can run it quickly many times without <tt>qsub</tt>
* Work in directory <tt>tiny</tt>
** <tt>ref.fa</tt>: 2 human proteins
** <tt>other.fa</tt>: a selected subset of proteins from 8 other mammalian species
** <tt>Makefile</tt>: a longer makefile
** <tt>brm.pl</tt>: a Perl script for finding ortholog groups and sorting them to directories

The <tt>Makefile</tt> runs the analysis in four stages. Stages 1,2 and 4 are done, you have to finish stage 3
* If you run <tt>make</tt> without argument, it will attempt to run all 4 stages, but stage 3 will not run, because it is missing
* Stage 1: run as <tt>make ref.brm</tt>
** It runs <tt>blast</tt> as in task A, then splits proteins into ortholog groups and creates one directory for each group with file <tt>prot.fa</tt> containing protein sequences
* Stage 2: run as <tt>make alignments</tt>
** In each directory with an ortholog group, it will create an alignment <tt>prot.phy</tt> and link it under names <tt>lg.phy</tt> and <tt>wag.phy</tt>
* Stage 3: run as <tt>make trees</tt> (needs to be written by you)
** In each directory with an ortholog group, it should create files <tt>lg.phy_phyml_tree</tt> and <tt>wag.phy_phyml_tree</tt> containing the results of the <tt>phyml</tt> program run with two different evolutionary models WAG and LG, where LG is the default
** Run <tt>phyml</tt> by commands of the form:<br><tt>phyml -i INPUT --datatype aa --bootstrap 0 --no_memory_check >LOG</tt><br><tt>phyml -i INPUT --model WAG --datatype aa --bootstrap 0 --no_memory_check >LOG</tt>
** Change <tt>INPUT</tt> and <tt>LOG</tt> in the commands to the appropriate filenames using <tt>make</tt> variables <tt>$@, $^, $*</tt> etc. The input should come from lg.phy or wag.phy in the directory of a gene and log should be the same as tree name with extension <tt>.log</tt> added (e.g. <tt>lg.phy_phyml_tree.log</tt>)
** Also add variables <tt>LG_TREES</tt> and <tt>WAG_TREES</tt> listing filenames of all desirable trees and uncomment phony target <tt>trees</tt> which uses these variables
* Stage 4:  run as <tt>make consensus</tt>
** Output trees from stage 3 are concatenated for each model separately to files <tt>lg/intree</tt>, <tt>wag/intree</tt> and then <tt>phylip</tt> is run to produce consensus trees <tt>lg.tree</tt> and <tt>wag.tree</tt>
** This stage also needs variables <tt>LG_TREES</tt> and <tt>WAG_TREES</tt> to be defined by you.

* Run your <tt>Makefile</tt> and check that the files <tt>lg.tree</tt> and <tt>wag.tree</tt> are produced
<!-- NOTEX -->
* '''Submit''' the whole directory <tt>tiny</tt>, including <tt>Makefile</tt> and all gene directories with tree files.
<!-- /NOTEX -->


===Task C (running make)===
* Copy your <tt>Makefile</tt> from part B to directory <tt>small</tt>, which contains 9 human proteins and run <tt>make</tt> on this slightly larger set
** Again, run it without <tt>qsub</tt>, but it will take some time, particularly if the server is busy 
* Look at the two resulting trees (<tt>wag.tree</tt>, <tt>lg.tree</tt>) using the <tt>figtree</tt> program
<!-- NOTEX -->
** it is available on vyuka, but you can also [https://github.com/rambaut/figtree/releases install it] on your computer if needed
<!-- /NOTEX -->
* In <tt>figtree</tt>, change the position of the root in the tree to make the opossum the outgroup (species branching as the first away from the others). This is done by clicking on opossum and thus selecting it, then pressing the Reroot button. 
* Also switch on displaying branch labels. These labels show for each branch of the tree, how many of the input trees support this branch. To do this, use the left panel with options. 
* Export the trees in pdf format as <tt>wag.tree.pdf</tt> and <tt>lg.tree.pdf</tt>
* Compare the two trees
** Note that the two children of each internal node are equivalent, so their placement higher or lower in the figure does not matter.
** Do the two trees differ? What is the highest and lowest support for a branch in each tree?
** Also compare your trees with the accepted "correct tree" found here http://genome-euro.ucsc.edu/images/phylo/hg38_100way.png (note that this tree contains many more species, but all ours are included)
<!-- NOTEX -->
** Write '''your observations to the protocol'''
* '''Submit''' the entire <tt>small</tt> directory (including the two pdf files)
<!-- /NOTEX -->

===Further possibilities===


<!-- NOTEX -->
Here are some possibilities for further experiments, in case you are interested
 (do not submit these):
<!-- /NOTEX -->
<!-- TEX
Here are some possibilities for further experiments, in case you are interested:
/TEX -->
* You could copy your extended <tt>Makefile</tt> to directory <tt>large</tt> and create trees for all ortholog groups in the big set
<!-- NOTEX -->
** This would take a long time, so submit it through qsub and only some time after the lecture is over to allow classmates to work on task A
<!-- /NOTEX -->
** After <tt>ref.brm</tt> si done, programs for individual genes can be run in parallel, so you can try running <tt>make -j 2</tt> and request 2 threads from <tt>qsub</tt>
* Phyml also supports other models, for example JTT  (see [http://www.atgc-montpellier.fr/download/papers/phyml_manual_2012.pdf manual]); you could try to play with those.
* Command <tt>touch FILENAME<tt> will change the modification time of the given file to the current time
** What happens when you run <tt>touch<tt> on some of the intermediate files in the analysis in task B? Does <tt>Makefile</tt> always run properly?

=Lpython=
<!-- NOTEX -->
[[#HWpython]]
<!-- /NOTEX -->

This lecture introduces the basics of the Python programming language. We will also cover basics of working with databases using the SQL language. 

<!-- NOTEX -->
The next three lectures
* Computer science students will use Python and SQLite3 and several advanced Python libraries for complex data processing
* Bioinformatics students will use several bioinformatics command-line tools
<!-- /NOTEX -->

==Overview, documentation==

'''Python'''
* Popular programming language
* Advantages: powerful language features, extensive libraries
* Disadvantages: interpreted language, can be slow 
* A very concise cheat sheet: [http://www.cogsci.rpi.edu/~destem/igd/python_cheat_sheet.pdf]
* A more detailed tutorial: [https://docs.python.org/3/tutorial/]

'''SQL'''
* Language for working with relational databases, more in a dedicated course
* We will cover basics of SQL and work with a simple DB system SQLite3 
* Typical database systems are complex, use server-client srchitecture. SQLite3 is a simple "database" stored in one file. You can think of SQLite3 not as a replacement for Oracle but as a replacement for <tt>fopen()</tt>.
* [https://www.sqlite.org/docs.html] SQLite3 documentation]
* [https://www.w3schools.com/sql/default.asp SQL tutorial]
* [[https://docs.python.org/3/library/sqlite3.html SQLite3 in Python documentation]


Outline of this lecture:
* We introduce a simple data set
* We look at several python scripts for processing this data set
* Solve task A, where you create another such a script
* We introduce basics of working with SQLite3 and writing SQL queries
* Solve tasks B1 and B2, where you write your own SQL queries
* We look at how to combine Python and SQLite
* Solve task C, where you write a program combining the two
* Students familiar with both Python and SQL may skip tasks A, B1, B2 and and do tasks C and D


==Dataset for this lecture==
* [https://www.imdb.com/ IMDb] is an online database of movies and TV series with user ratings
* We have downloaded a preprocessed dataset of selected TV series ratings from [https://github.com/nazareno/imdb-series/ GitHub]
* From this dataset, we have selected only 10 series with the highest average number of voting users
* Data are two files in csv format: list of series, list of episodes

File <tt>series.cvs</tt> contains one row per series 
* Columns: (0) series id, (1) series title, (2) TV channel:
<pre>
3,Breaking Bad,AMC
2,Sherlock,BBC
1,Game of Thrones,HBO 
</pre>

File <tt>episodes.csv</tt> contains one row per episode:
* Columns: (0) series id, (1) episode title, (2) episode order within the whole series, (3) season number, (4) episode number within season, (5) user rating, (6) the number of votes
* Here is a sample of 4 episodes from the Game of Thrones series
* If the episode title contains a comma, the whole title is in quotation marks
<pre>
1,"Dark Wings, Dark Words",22,3,2,8.6,12714
1,No One,58,6,8,8.3,20709
1,Battle of the Bastards,59,6,9,9.9,138353
1,The Winds of Winter,60,6,10,9.9,93680
</pre>

==Several Python scripts==

We will illustrate basic features of Python on several scripts working with these files.

<!-- TODO short description of python, more detailed description of the scripts -->

===prog1.py=== 
Print the second column (series title) from <tt>series.csv</tt>
<pre>
#! /usr/bin/python3

# open a file for reading
with open('series.csv') as csvfile:
    # iterate over lines of the input file
    for line in csvfile:
        # split a line into columns at commas
        columns = line.split(",")
        # print the second column
        print(columns[1])
</pre>

===prog2.py=== 
Print the list of series of each TV channel
* For illustration we also separately count the series for each channel, but the count could be obtained as the length of the list
* For simplicity we use library data structure <tt>defaultdict</tt> instead of plain python dictionary
<pre>
#! /usr/bin/python3
from collections import defaultdict

# Create a dictionary in which default value
# for non-existent key is 0 (type int)
# For each channel we will count the series
channel_counts = defaultdict(int)

# Create a dictionary for keeping a list of series per channel
# default value empty list
channel_lists = defaultdict(list)

# open a file and iterate over lines
with open('series.csv') as csvfile:
    for line in csvfile:
        # strip whitespace (e.g. end of line) from end of line
        line = line.rstrip()
        # split line into columns, find channel and series names
        columns = line.split(",")
        channel = columns[2]
        series = columns[1]
        # increase counter for channel
        channel_counts[channel] += 1
        # add series to list for the channel
        channel_lists[channel].append(series)

# print counts
print("Counts:")
for channel in channel_counts:
    print("The number of series for channel \"%s\" is %d" 
    % (channel, channel_counts[channel]))


# print series lists
print("\nLists:")
for channel in channel_lists:
    list = ", ".join(channel_lists[channel]) 
    print("series for channel \"%s\": %s" % (channel,list))
</pre>

===prog3.py=== 
Find the episode with the highest number of votes among all episodes
* We use a library for csv parsing to deal with quotation marks.
<pre>
#! /usr/bin/python3
import csv

#keep maximum number of votes and its episode
max_votes = 0
max_votes_episode = None

# open a file
with open('episodes.csv') as csvfile:
    # create a reader for parsing csv files
    reader = csv.reader(csvfile, delimiter=',', quotechar='"')
    # iterate over rows already split into columns
    for row in reader:
        votes = int(row[6])
        if votes > max_votes:
            max_votes = votes
            max_votes_episode = row[1]
        
# print result
print("Maximum votes %d in episode \"%s\"" % (max_votes, max_votes_episode))
</pre>

===prog4.py=== 
Example of function definition, reading the whole file into a 2d array
<pre>
#! /usr/bin/python3
import csv

def read_csv_to_list(filename):
    # create empty list
    rows = []
    # open a file
    with open(filename) as csvfile:
        # create a reader for parsing csv files
        reader = csv.reader(csvfile, delimiter=',', quotechar='"')
        # iterate over rows already split into columns
        for row in reader:
            rows.append(row)
    return rows

series = read_csv_to_list('series.csv')
episodes = read_csv_to_list('episodes.csv')
print("the number of episodes is %d" % len(episodes))
# further processing of series and episodes...
</pre>

'''Now do [[#HWpython]], task A'''

==SQL and SQLite==

===Creating a database===
SQLite3 database is a file with your data stored in some special format. To load our csv file to a SQLite database, run command:
<pre>
sqlite3 series.db < create_db.sql
</pre>

Contents of <tt>create_db.sql</tt> file:
<pre>
CREATE TABLE series (
  id INT,
  title TEXT,
  channel TEXT
);
.mode csv
.import series.csv series
CREATE TABLE episodes (
  seriesId INT,
  title TEXT,
  orderInSeries INT,
  season INT,
  orderInSeason INT,
  rating REAL,
  votes INT
);
.mode csv
.import episodes.csv episodes
</pre>
* The two <tt>CREATE TABLE</tt> commnds create two tables naamed <tt>series</tt> and <tt>episodes</tt>
* For each column (attribute) of the table we list if name and type.
* Commands starting with a dot are special SQLite3 commands, not part of SQL itself. Command <tt>.import</tt> reads the txt file and stores it in a table.

Other useful SQLite3 commands;
* <tt>.schema tableName</tt> (lists columns of a given table)
* <tt>.mode column</tt> and <tt>.headers on</tt> (turn on human-friendly formatting (not good for further processing)

===SQL queries===
* Run <tt>sqlite3 series.db</tt> to get an SQLite command-line where you can interact with your database
* Then type the following queries which illustrate the basic features of SQL
<pre>
/*  switch on human-friendly formatting */
.mode column
.headers on

/* print title of each series (as prog1.py) */
SELECT title FROM series;

/* sort titles alphabetically */
SELECT title FROM series ORDER BY title;

/* find the highest vote number among episodes */
SELECT MAX(votes) FROM episodes;

/* find the episode with the highest number of votes, as prog3.py */
SELECT title, votes FROM episodes
  ORDER BY votes DESC LIMIT 1;

/* print all episodes with at least 50k votes, order by votes */
SELECT title, votes FROM episodes
  WHERE votes>50000 ORDER BY votes desc;

/* join series and episodes tables, print 10 episodes
 * with the highest number of votes */
SELECT s.title, e.title, votes
  FROM episodes AS e, series AS s
  WHERE e.seriesId=s.id
  ORDER BY votes desc limit 10;

/* compute the number of series per channel, as prog2.py */
SELECT channel, COUNT() as series_count
  FROM series GROUP BY channel;

/* print the number of episodes and average rating per season and series */
SELECT seriesId, season, COUNT() AS episode_count, AVG(rating) AS rating
  FROM episodes GROUP BY seriesId, season;
</pre>

'''Now do [[#HWpython]], tasks B1, B2'''

==Accessing a database from Python==

We will use sqlite3 library for Python to access data from the database and to process in further in the Python program.

===read_db.py===
* The following script illustrates running a SELECT query and the getting results
<pre>
#! /usr/bin/python3
import sqlite3

# connect to a database 
connection = sqlite3.connect('series.db')
# create a "cursor" for working with the database
cursor = connection.cursor()

# run a select query
# supply parameters of the query using placeholders ?
threshold = 40000
cursor.execute("""SELECT title, votes FROM episodes
  WHERE votes>? ORDER BY votes desc""", (threshold,))

# retrieve results of the query
for row in cursor:
    print("Episode \"%s\" votes %s" % (row[0],row[1]))
    
# close db connection
connection.close()
</pre>

===write_db.py===
This script illustrates creating a new database containing a multiplication table
<pre>
#! /usr/bin/python3
import sqlite3

# connect to a database 
connection = sqlite3.connect('multiplication.db')
# create a "cursor" for working with the database
cursor = connection.cursor()

cursor.execute("""
CREATE TABLE mult_table (
a INT, b INT, mult INT)
""")

for a in range(1,11):
    for b in range(1,11):
        cursor.execute("INSERT INTO mult_table (a,b,mult) VALUES (?,?,?)",
                       (a,b,a*b))

# important: save the changes
connection.commit()
    
# close db connection
connection.close()
</pre>

We can check the result by running command
<pre>
sqlite3 multiplication.db "SELECT * FROM mult_table;"
</pre>

'''Now do [[#HWpython]], task C'''

==HWpython==

<!-- NOTEX -->
See also the [[#Lpython|lecture]]
<!-- /NOTEX -->

===Introduction===
Choose one of the options:
* Tasks A, B1, B2, C (recommended for beginners)
* Tasks C, D (recommended for experienced Python/SQL programmers)

===Preparation===
Copy files:
<pre>
mkdir python
cd python
cp -iv /tasks/python/* .
</pre>

The directory contains the following files:
* <tt>*.py</tt>: python scripts from the lecture, included for convenience
* <tt>series.csv</tt>, <tt>episodes.csv</tt>: data files introduced in the lecture
* <tt>create_db.sql</tt>: SQL commands to create the database needed in tasks B1, B2, C, D
<!-- NOTEX -->
* <tt>protocol.txt</tt>: fill in and submit the protocol. 
<!-- /NOTEX -->

===Task A (Python)===
Write a script <tt>taskA.py</tt> which reads both csv files and outputs for each TV channel the total number of episodes in their series combined. Run your script as follows:
<pre>
./taskA.py > taskA.txt
</pre>
One of the lines of your output should be:
<pre>
The number of episodes for channel "HBO" is 76
</pre>
<!-- NOTEX -->
'''Submit''' file <tt>taskA.py</tt> with your script and the output file <tt>taskA.txt</tt>:
<!-- /NOTEX -->

Hints:
* A good place to start is <tt>prog4.py</tt> with reading both csv files and <tt>prog2.py</tt> with a dictionary of counters
* It might be useful to build a dictionary linking the series id to the channel name for that series

===Task B1 (SQL)===
To prepare the database for tasks B1, B2, C and D, run the command:
<pre>
sqlite3 series.db < create_db.sql
</pre>

To verify that your database was created correctly, you can run the following commands:
<pre>
sqlite3 series.db ".tables"
# output should be  episodes  series  

sqlite3 series.db "select count() from episodes; select count() from series;"
# output should be 348 and 10
</pre>

The [[#Lpython#SQL_queries|last query in the lecture]] counts the number of episodes and average rating per each season of each series:
<pre>
SELECT seriesId, season, COUNT() AS episode_count, AVG(rating) AS rating
  FROM episodes GROUP BY seriesId, season;
</pre>
Use join with the <tt>series</tt> table to replace the numeric series id with the series title and add the channel name. Write your SQL query to file <tt>taskB1.sql</tt>. The first two lines of the file should be
<pre>
.mode column
.headers on
</pre>
Run your query as follows:
<pre>
sqlite3 series.db < taskB1.sql > taskB1.txt
</pre>
For example, both seasons of True Detective by HBO have 8 episodes and average ratings 9.3 and 8.25
<pre>
True Detective   HBO         1           8              9.3       
True Detective   HBO         2           8              8.25      
</pre>
<!-- NOTEX -->
'''Submit'''  <tt>taskB1.sql</tt> and <tt>taskB1.txt</tt>
<!-- /NOTEX -->

===Task B2 (SQL)===
For each channel compute the total count and average rating of all their episodes. Write your SQL query to file <tt>taskB2.sql</tt>. As before, the first two lines of the file should be
<pre>
.mode column
.headers on
</pre>
Run your query as follows:
<pre>
sqlite3 series.db < taskB2.sql > taskB2.txt
</pre>
For example, all 76 episodes for the two HBO series have average rating as follows:
<pre>
HBO         76          8.98947368421053
</pre>
<!-- NOTEX -->
'''Submit'''  <tt>taskB2.sql</tt> and <tt>taskB2.txt</tt>
<!-- /NOTEX -->

===Task C (Python+SQL)===
If you have not done so already, create an SQLite database, as explained at the beginning of [[#Task_B1 (SQL)|task B1]].

Write a Python script that runs the last query from the lecture (shown below) and stores its results in a separate table called <tt>seasons</tt> in the <tt>series.db</tt> database file
<pre>
/* print the number of episodes and average rating per season and series */
SELECT seriesId, season, COUNT() AS episode_count, AVG(rating) AS rating
  FROM episodes GROUP BY seriesId, season;
</pre>
* SQL can store the results from a query directly in a table, but in this task you should instead read each row of the <tt>SELECT</tt> query in Python and to store it by running <tt>INSERT</tt> command from Python
* Also do not forget to create the new table in the database with appropriate column names and types. You can execute <tt>CREATE TABLE</tt> command from Python
* The cursor from the <tt>SELECT</tt> query is needed while you iterate over the results. Therefore create two cursors - one for reading the database and one for writing.
* If you change your database during debugging, you can start over by running the command for creating the database above
* Store the script as <tt>taskC.py</tt>. 

To check that your table was created, you can run command 
<pre>
sqlite3 series.db "SELECT * FROM seasons;"
</pre>
This will print many lines, including this one: <tt>"5|1|8|9.3"<tt> which is for season 1 of series 5 (True Detective).

<!-- NOTEX -->
'''Submit''' your script <tt>taskC.py</tt> and the modified database <tt>series.db</tt>.
<!-- /NOTEX -->

===Task D (SQL, optionally Python)===
For each pair of consecutive seasons within each series, compute how much has the average rating increased or decreased.
* For example in the Sherlock series, season 1 had rating 8.825 and season 2 rating 9.26666666666667, and thus the difference in ratings is 0.44166666666667
* Print a table containing series name, season number x, average rating in season x and average rating in season x+1
* The table should be ordered by the difference between the last two columns, i.e. from seasons with the highest increase to seasons to the highest drop.
* One option is to run a query in SQL in which you join the <tt>seasons</tt> table from task C with itself and select rows that belong to the same series and successive seasons
* You can also read the rows of the <tt>seasons</tt< table in Python, combine information from rows for successive seasons of the same series and create the final report by sorting
<!-- NOTEX -->
* '''Submit''' your code as <tt>taskD.py</tt> or <tt>taskD.sql</tt> and the resulting table as <tt>taskD.txt</tt>
<!-- /NOTEX -->

The output should start like this (the formatting may differ):
<pre>
series      season x    rating for x  rating for x+1  
----------  ----------  ------------  ----------------
Sherlock    1           8.825         9.26666666666667
Breaking B  4           9.0           9.375           
</pre>

When using SQL without Python, include the following two lines in <tt>taskD.sql</tt>
<pre>
.mode column
.headers on
</pre>
and run your query as <tt>sqlite3 series.db < taskD.sql > taskD.txt</tt>

=Lweb=
<!-- NOTEX -->
[[#HWweb]]
<!-- /NOTEX -->

In this lecture, we will extract information from a website using Python and existing Python libraries. We will store the results in an SQLite database. These results will be analyzed further in the following lectures.

== Scraping webpages ==

In Python, the simplest tool for scraping webpages is <tt>[https://docs.python.org/2/library/urllib2.html urllib2]</tt> library. Example usage:
<pre>
import urllib2
f = urllib2.urlopen('http://www.python.org/')
print f.read()
</pre>

You can also use <tt>requests</tt> package:
<pre>
import requests
r = requests.get("http://en.wikipedia.org")
print(r.text[:10])
</pre>

== Parsing webpages ==

We will use <tt>beautifulsoup4</tt> library for parsing HTML.
* In your code, we recommend following the examples at the beginning of the [http://www.crummy.com/software/BeautifulSoup/bs4/doc/ documentation] and the example of [http://www.crummy.com/software/BeautifulSoup/bs4/doc/#css-selectors CSS selectors].
* Information you need to extract is located within the structure of the HTML document
* To find out, how is the document structured, use Inspect element feature in Chrome (right click on the text of interest within the website)
* Based on this information, create a CSS selector

== Parsing dates ==

To parse dates (written as a text), you have two options:
* <tt>[https://docs.python.org/3/library/datetime.html#strftime-and-strptime-behavior datetime.strptime]</tt> 
* <tt>[https://dateutil.readthedocs.org/en/latest/parser.html dateutil]</tt> package.

== Other usefull tips ==
* Don't forget to commit to your SQLite3 database (call <tt>db.commit()</tt>).
* SQL command <tt>CREATE TABLE IF NOT EXISTS</tt> can be usefull at the start of your script.
* Use <tt>screen</tt> command for long running scripts.
* All packages are installed on our server. If you use your own laptop, you need to install them using <tt>pip</tt> (preferably in an  <tt>virtualenv</tt>).

=HWweb=
<!-- NOTEX -->
See [[#Lweb|the lecture]]
<!-- /NOTEX -->

<!-- NOTEX -->
Submit by copying requested files to <tt>/submit/web/username/</tt>
<!-- /NOTEX -->

'''General goal:''' Scrape comments from user discussions at the <tt>sme.sk</tt> website. Store comments  from several (hundreds) users from the last month in an SQLite3 database.

==Task A==

Create SQLite3 "database" with appropriate schema for storing comments from SME.sk discussions.
You will probably need tables for users and comments. You don't need to store which comment replies to which one but store the date and time when the comment was made.

<!-- NOTEX -->
Submit two files:
* <tt>db.sqlite3</tt> - the database
* <tt>schema.txt</tt> - a brief description of your schema and rationale behind it
<!-- /NOTEX -->


==Task B==

Build a crawler, which crawls comments in sme.sk discussions.
You have two options:
* For fewer points: Script which gets URL of a user (e.g. http://ekonomika.sme.sk/diskusie/user_profile.php?id_user=157432) and crawls his comments from the last month.
* For more points: Scripts which gets one starting URL (either user profile or some discussion, your choice) and automatically discovers users and crawls their comments.

This crawler should store the comments in SQLite3 database built in the previous task.

<!-- NOTEX -->
Submit the following:
* <tt>db.sqlite3</tt> - the database
* every python script used for crawling
* <tt>README</tt> (how to start your crawler)
<!-- /NOTEX -->

=Lflask=
<!-- NOTEX -->
[[#HWflask]]
<!-- /NOTEX -->

In this lecture, we will use Python to process user comments obtained in the previous lecture. 
* We will display information about individual users as a dynamic website written in Flask frmework
* We will use simple text processing utilities from ScikitLearn library to extract word use statistics from the comments

==Flask==

[http://flask.pocoo.org/docs/1.0/quickstart/ Flask] is a simple web server for Python. Using Flask you can write a simple dynamic website in Python. 


===Running Flask===

You can find a sample Flask application at <tt>/tasks/flask/simple_flask.</tt> Run it using these commands:
<pre>
cd <your directory>
export FLASK_APP=main.py
export FLASK_ENV=development # this is optional, but recommended for debugging

# before running the following, change the port number
# so that no two users use the same number
flask run --port=4247 
</pre>

Flask starts a webserver and serves the pages created in your Flask application,. Keep it running while you need to access these pages. 

To view these pages, open a web browser on the same computer where the Flask is running, e.g. <tt>chromium-browser  http://localhost:4247/</tt> (change the port number to the one used in Flask). If you want to run graphical applications, such as Chromium, from our server, do not forget <tt>-XC</tt> switch in <tt>ssh</tt>.

<!-- TODO nefunguje --> 
You can also run Flask on a server but the browser at your own computer. Just replace <tt>localhost</tt> in the URL with the server address. However, firewalls may prevent you from accessing strange ports. You can get around by creating a proxy: 
* On your local machine create a SOCKS proxy server on port 8000 using command<br><tt>ssh username@server_addess -D 8000</tt>
* keep this SSH session open while working
* Set the HTTP proxy in your browser to localhost with port 8000. Then all web traffic goes through server via the SSH tunnel.  


===Structure of a Flask application===

* The provided Flask application resides in the <tt>main.py</tt> script. 
* Some functions in this script are annotated with decorators starting with <tt>@app</tt>.
* Decorator <tt>@app.before_request</tt> marks a function which will be executed before processing a particular request from a web browser. In this case we open a database connection and store it in a special variable <tt>g</tt> which can be used to store variables for a particular request.
* Decorator <tt>@app.route('/')</tt> marks a function which will serve the main  page of the application with URL <tt>http://localhost:4247/</tt>. Similarly decorator <tt>@app.route('/wat/<random_id>/')</tt> marks a function which will serve URLs of the form <tt>http://localhost:4247/wat/100</tt> where the particular string which the user uses in the URL (here <tt>100</tt>) will be stored in <tt>random_id</tt> variable accessible within the function.
* Functions serving a request return a string containing the requested webpage (typically a HTML document). For example, function <tt>wat</tt> returns a simple string without any HTML markup.
* To more easily construct a full HTML document, you can use [http://jinja.pocoo.org/docs/dev/templates/ jinja2]  templating language, as is done in the <tt>home</tt> function. The template itself is in file <tt>templates/main.html</tt>.


==Processing text==

The main tool we will use for processing text is [http://scikit--learn.org/stable/modules/generated/sklearn.feature_extraction.text.CountVectorizer.html <tt>CountVectorizer</tt>] class from the Scikit-learn library.
It transforms a text into a bag of words representation. In this representation we get the list of word and the count for each word. Example:

<pre>
from sklearn.feature_extraction.text import CountVectorizer

vec = CountVectorizer(strip_accents='unicode')

texts = [
 "Ema ma mamu.",
 "Zirafa sa vo vani kupe a hneva sa."
]

t = vec.fit_transform(texts).todense()

print(t)
# prints:
# [[1 0 0 1 1 0 0 0 0]
#  [0 1 1 0 0 2 1 1 1]]

print(vec.vocabulary_)
# prints:
# {'vani': 6, 'ema': 0, 'kupe': 2, 'mamu': 4, 
# 'hneva': 1, 'sa': 5, 'ma': 3, 'vo': 7, 'zirafa': 8}
</pre>

==NumPy arrays==

Array <tt>t</tt> in the example above is a NumPy array provided by the [https://numpy.org/ NumPy library]. This librray has also lots of nice tricks. First lets create two matrices:
<pre>
>>> import numpy as np
>>> a = np.array([[1,2,3],[4,5,6]])
>>> b = np.array([[7,8],[9,10],[11,12]])
>>> a
array([[1, 2, 3],
       [4, 5, 6]])
>>> b
array([[ 7,  8],
       [ 9, 10],
       [11, 12]])
</pre>

We can sum these matrices or multiply them by some number:
<pre>
>>> 3 * a
array([[ 3,  6,  9],
       [12, 15, 18]])
>>> a + 3 * a
array([[ 4,  8, 12],
       [16, 20, 24]])
</pre>

We can calculate sum of elements in each matrix, or sum by some axis:
<pre>
>>> np.sum(a)
21
>>> np.sum(a, axis=1)
array([ 6, 15])
>>> np.sum(a, axis=0)
array([5, 7, 9])
</pre>

There are many other useful functions, check [https://docs.scipy.org/doc/numpy-dev/user/quickstart.html the documentation].

=HWflask=

<!-- NOTEX -->
See [[#Lflask|the lecture]]
<!-- /NOTEX -->

'''General goal:'''
Build a simple website, which lists all crawled users and for each users has a page with simple statistics regarding the posts of this user.


<!-- NOTEX -->
Submit your source code (web server and preprocessing scripts) and database files. Copy these files to <tt>/submit/flask/username/</tt>
<!-- /NOTEX -->

<!-- NOTEX -->
This lesson requires crawled data from previous lesson, if you don't have one, you can find it at (and thank Baska): <tt>/tasks/flask/db.sqlite3</tt>
<!-- /NOTEX -->

==Task A==

Create a simple Flask web application which:
* Has a homepage with is a list of all users (with links to their pages).
* Has a page for each user with basic information: the nickname, the number of posts and the last 10 posts of this user.

==Task B==

For each user preprocess and store 
* the list 10 most frequently used words 
* the list of top 10 words typical for this user (words which this user uses much more often than other users, come up with some simple heuristics for measuring this)
Show this information on the page of each user.

Hint: To get the most frequently used words for each user, you can use 
[http://docs.scipy.org/doc/numpy/reference/generated/numpy.argsort.html#numpy.argsort argsort from NumPy.]


==Task C==

Preprocess and store the list of top three similar users for each user (try to come up with some simple definition of similarity based on the text in the posts). Again show this information on the user page.

'''Bonus:''' Try to use some simple topic modeling (e.g. PCA as in TruncatedSVD from scikit-learn) and use it for finding similar users.

=Ljavascript=
<!-- NOTEX -->
[[#HWjavascript]]
<!-- /NOTEX -->


In this lecture we will extend the website from the previous lecture with interactive visualizations written in JavaScript. We will not cover details of the JavaScript programming language, only use visualization contained in the Google Charts library.

Your goal is to take examples from [https://developers.google.com/chart/interactive/docs/ the documentation] and tweak them for your purposes.

'''Tips:'''
* You can write your data into JavaScript data structures in a Flask template. It is a bad practice, but sufficient for this lecture. (A better way is to load data in JSON format through API).
* Consult the [[#Lflask|previous lecture]] on running and accessing Flask applications.

=HWjavascript=

<!-- NOTEX -->
See [[#Ljavascript|the lecture]]
<!-- /NOTEX -->

'''General goal:''' Extend the user pages from the previous lecture with simple visualizations.

<!-- NOTEX -->
Submit your source code to <tt>/submit/javascript/username/</tt>
<!-- /NOTEX -->

==Task A==

Display a calendar, which shows during which days was the user active. Use [https://developers.google.com/chart/interactive/docs/gallery/calendar calendar from Google Charts].

==Task B==

Show a histogram of comment lengths. Use [https://developers.google.com/chart/interactive/docs/gallery/histogram histogram from Google Charts].

==Task C==

Show a word tree for a user using [https://developers.google.com/chart/interactive/docs/gallery/wordtree word tree from Google Charts]. Try to normalize the text before building the tree(convert to lowercase, remove accents). <tt>CountVectorizer</tt> has <tt>build_analyzer</tt> method, which returns a function, which does this for you.


=Lbioinf1=
<!-- NOTEX -->
[[#HWbioinf1]]
<!-- /NOTEX -->

The next three lectures at targeted at the students in the Bioinformatics program and the goal is to get experience with several common bioinformatics tools. Students will learn more about the algorithms and models behind these tools in the [http://compbio.fmph.uniba.sk/vyuka/mbi/index.php/%C3%9Avod Methods in bioinformatics] course.

==Overview of DNA sequencing and assembly==
'''DNA sequencing''' is a technology of reading the order of nucleotides along a DNA strand 
* The result is represented as a string of A,C,G,T
* Only fragments of DNA of limited length can be read, these are called '''sequencing reads'''
* Different technologies produce reads of different characteristics
* Examples:
** '''Illumina sequencers''' produce short reads (typical length 100-200bp), but in great quantities and very low error rate (<0.1%) 
** Illumina reads usually come in '''pairs''' sequenced from both ends of a DNA fragment of an approximately known length
** '''Oxford nanopore sequencers''' produce longer reads (thousands of basepairs  or more), but the error rates are higher (10-15%) 


The goal of '''genome sequencing''' is to read all chromosomes of an organism
* Sequencing machines produce many reads coming from different parts of the genome
* Using software tools called '''sequence assemblers''', these reads are glued together based on overlaps
* Ideally we would get the true chromosomes, but often we get only shorter fragments called '''contigs'''
* The results of assembly can contain errors
* We prefer longer contigs with fewer errors

==Sequence alignments and dotplots==
* '''Sequence alignment''' is the task of finding similarities between DNA (or protein) sequences 
* Here is an example - short similarity between region at positions 344,447..344,517 of one sequence and positions 3,261..3,327 of another sequence
<pre>
Query: 344447 tctccgacggtgatggcgttgtgcgtcctctatttcttttatttctttttgttttatttc 344506
              |||||||| |||||||||||||||||| ||||||| |||||||||||| ||   ||||||
Sbjct: 3261   tctccgacagtgatggcgttgtgcgtc-tctatttattttatttctttgtg---tatttc 3316

Query: 344507 tctgactaccg 344517
              |||||||||||
Sbjct: 3317   tctgactaccg 3327
</pre>

* Alignments can be stored in many formats and visualised as dotplots
* In a '''dotplot''', the x-axis correspond to positions in one sequence and the y-axis in another sequence
* Diagonal lines show alignments between the sequences (direction of the diagonal shows which DNA strand was aligned)

[[Image:Dotplot-mt-human-dros.png|center|thumb|250px|Dotplot of human and ''Drosophila'' mitochondrial genomes]]

==File formats==

===Fasta===
* Fasta is a format for storing DNA, RNA and protein sequences 
* We have already seen Fasta files in [[#HWperl|Perl exercises]]
* Each sequence is given on several lines of the file. The first line starts with ">" followed by an identifier of the sequence and optionally some further description separated by whitespace
* The sequence itself is on the second line; long sequences are split into multiple lines
<pre>
>SRR022868.1845_1
AAATTTAGGAAAAGATGATTTAGCAACATTTAGCCTTAATGAAAGACCAGATTCTGTTGCCATGTTTGAATGCCTTAAACCAGTAGCAGAATCAGTATAAA
>SRR022868.1846_1
TAGCGTTGTAAAATAAATTTCTAGAATGGAAGTGATGATATTGAAATACACTCAGATCCTGAATGAAAGATTTATTAAAGTTAAGACGAGAGTCTCATTAT
</pre>

===Fastq===
* Fastq is a format for storing sequencing reads, containing DNA sequences but also quality information about each nucleotide 
* More in the [[#Lperl#The_second_input_file_for_today:_DNA_sequencing_reads_.28fastq.29|lecture on Perl]]

===Sam/Bam===
* Sam and Bam are formats for storing alignments of sequencing reads (or other sequences) to a genome 
* For each read, the file contains the read itself, its quality, but also the chromosome/contig name and position where this read is likely coming from, and an additional information e.g. about mapping quality (confidence in the correct location)
* Sam files are text-based, thus easier to check manually; Bam files are binary and compressed, thus smaller and faster to read
* We can easily convert between Sam and Bam using [https://github.com/samtools/samtools samtools]
* [https://samtools.github.io/hts-specs/SAMv1.pdf Full documentation of the format]

===Paf format===
* Another format for storing alignments used in the minimap2 tool 
* [https://github.com/lh3/miniasm/blob/master/PAF.md Full documentation of the format]

===Gzip===
* Gzip is a general-purpose tool for file compression
* It is often used in bioinformatics on large Fastq or Fasta files
* Running command <tt>gzip filename.ext</tt> will create compressed file <tt>filename.ext.gz</tt> (original file will be deleted). 
* The reverse process is done by <tt>gunzip filename.ext.gz</tt>
* However, we can access the file withou uncompressining it. Command <tt>zcat filename.ext.gz</tt> prints the content of a gzipped file and keep the gzipped file as is. We can use pipe to do ruther processing on the file.
* To manually page through the content of a gzipped file use <tt>zless filename.ext.gz</tt>
* Some bioinformatics tools can work directly with gzipped files

=HWbioinf1=

<!-- NOTEX -->
See also the [[#Lbioinf1|lecture]]

Submit the protocol and the required files to <tt>/submit/bioinf1</tt>
<!-- /NOTEX -->

==Task A: examine input files==
Copy files from <tt>/tasks/bioinf1/</tt> as follows:
<pre>
mkdir bioinf1
cd bioinf1
cp -iv /tasks/bioinf1/* .
</pre>
* <tt>ref.fasta</tt> is a piece of genome from ''Escherichia coli'' 
* <tt>miseq_R1.fastq.gz</tt> and <tt>miseq_R2.fastq.gz</tt> are sequencing reads from Illumina MiSeq sequencer. First reads in pairs are in the R1 file, second reads in the R2 file. These reads come from the region in <tt>ref.fasta</tt>
* <tt>nanopore.fasta</tt> are nanopore sequencing reads in FASTA format (without qualities). These reads are also from the region in <tt>ref.fasta</tt>

<!-- !! -->
Try to find the answers to the following questions using command-line tools. 
<!-- NOTEX -->
In your '''protocol''', note down the '''commands''' as well as the '''answers'''.
<!-- /NOTEX -->

(a) How many reads are in the MiSeq files? Is the number of reads the same in both files? 
* Try command <tt>zcat miseq_R1.fastq.gz | wc -l </tt>
* Can you figure out the answer from the result of this command?

(b) How long are individual reads in the miseq files?
* Look at the file using <tt>zless</tt> - do all reads appear to be of an equal length?
* Extend the following command with <tt>tail</tt> and <tt>wc -c</tt> to get the length of the first read: <tt>zcat miseq_R1.fastq.gz | head -n 2</tt>
* Do not forget to consider the end of the line character
* Repeat for both MiSeq files

(c) How many reads are in the nanopore file (beware - different format)

(d) What is the average length of the reads in the nanopore file?
* Try command: <tt>samtools faidx nanopore.fasta</tt>
* This creates <tt>nanopore.fasta.fai</tt> file, where the second column contains sequence length of each read
* Compute the average of this column by a one-liner: <tt>perl -lane '$s+=$F[1]; $n++; END { print $s/$n }' nanopore.fasta.fai</tt>

(e) How long is the sequence in the <tt>ref.fasta</tt> file?

==Task B: assemble the sequence from the reads==
* We will pretend that the correct answer (<tt>ref.fasta</tt>) is not known and we will try to assemble it from the reads
* We will assemble Illumina reads by program SPAdes and nanopore reads by miniasm
* Assembly takes several minutes, we will run it in the background using <tt>screen</tt> command

SPAdes
* Run <tt>screen -S spades</tt>
* Press Enter to get command-line, then run the follwing command:<br>
:: <tt>spades.py -t 1 -m 1 --pe1-1 miseq_R1.fastq.gz --pe1-2 miseq_R2.fastq.gz -o spades > spades.log</tt>
* Press <tt>Ctrl-a</tt> followed by <tt>d</tt>
* This will take you out of <tt>screen</tt> command
* Run <tt>top</tt> command to check that your command is running

Minimap
* Create file <tt>minimap.sh</tt> containing the following commands:
<pre>
# Find alignments between pairs of reads
minimap2 -x ava-ont -t 1 nanopore.fasta nanopore.fasta | gzip -1 > nanopore.paf.gz 
# Use overlaps to compute the assembled genome
miniasm -f nanopore.fasta nanopore.paf.gz > miniasm.gfa 2> miniasm.log
# Convert genome to fasta format
perl -lane 'print ">$F[1]\n$F[2]" if $F[0] eq "S"' miniasm.gfa > miniasm.fasta
# Align reads to the assembled genome
minimap2 -x map-ont --secondary=no -t 1 miniasm.fasta nanopore.fasta | gzip -1 > miniasm.paf.gz
# Polish the genome by finding consensus of aligned reads at each position
racon -t 1 -u nanopore.fasta miniasm.paf.gz miniasm.fasta > miniasm2.fasta
</pre>
* Run <tt>screen -S minimap</tt>
* In screen, run <tt>source ./minimap.sh</tt>
* Press <tt>Ctrl-a d</tt> to exit <tt>screen</tt>


To check if your commands have finished:
* Re-enter the screen environment using <tt>screen -r spades</tt> or <tt>screen -r miniasm</tt>
* If the command finished, terminate <tt>screen</tt> by pressing <tt>Ctrl-d</tt> or typing <tt>exit</tt>

Examine the outputs. 
<!-- NOTEX -->
Write '''commands''' and '''answers''' to your '''protocol'''.
<!-- /NOTEX -->
* Copy output of SPAdes under a new filename: <tt>cp -ip spades/contigs.fasta spades.fasta</tt>
* Output of miniasm should be in <tt>miniasm2.fasta</tt>
* (a) How many contigs are in each of these two files?
* (b) What can you find out from the names of contigs in <tt>spades.fasta</tt>? What is the length of the shortest and longest contigs? Cov in the names is abbreviation of read coverage - the average number of reads covering a position on the contig. Do the reads have similar coverage, or are there big differences? 
** Use command <tt>grep '>' spades.fasta</tt>
* (c) What are the lengths of contigs in <tt>miniasm2.fa</tt> file? (you can use <tt>LN:i:</tt> in the name of contigs)

<!-- NOTEX -->
'''Submit files''' <tt>miniasm2.fasta</tt> and <tt>spades.fasta</tt>
<!-- /NOTEX -->

==Task C: compare assemblies using Quast command==
* We have found basic characteristics of the two assemblies in task B
* Now we will use program Quast to compare both assemblies to the correct answer in <tt>ref.fa</tt>
<pre>
quast.py -R ref.fasta miniasm2.fasta spades.fasta -o stats
</pre>
* Submit file <tt>stats/report.txt</tt>

Look at the results in <tt>stats/report.txt</tt> and '''answer''' the following questions in your '''protocol''':
* (a) How many contigs quast reported in the two assemblies? Does it agree with your counts in part B?
* (b) What is the number of mismatches per 100kb in the two assemblies? Which one is better? Why do you think it is so? (look at the properties of used sequencing technologies in the [[#Lbioinf1#Overview_of_DNA_sequencing_and_assembly|lecture]])
* (c) What portion of the reference sequence is covered by the two assemblies (genome fraction)? Which assembly is better in this aspect?
* (d) What is the length of the longest alignment between contigs and the reference in the two assemblies? Which assembly is better in this aspect?

==Task D: create dotplots of assemblies==
* We will now visualize alignments between each assembly and the reference genome using dotplots
** As in other tasks, write '''commands''' and '''answers''' to your '''protocol'''

* (a) Create dotplot comparing miniasm assembly to the reference sequence
<pre>
# alignments
minimap2 -x asm10 -t 1 ref.fasta miniasm2.fasta > ref-miniasm2.paf
# creating dotplot
/usr/local/share/miniasm/miniasm/minidot -f 12 ref-miniasm2.paf | ps2pdf -dEPSCrop - ref-miniasm2.pdf
# displaying dotplot - if this does not work, copy the pdf file to your commputer and view there
evince ref-miniasm2.pdf &
</pre>
** x-axis is reference, y-axis assembly
** Which part of the reference is missing in the assembly?
** Do you see any other big differences between the assembly and the reference?

* (b) Use analogous commands to create dotplot for spades assembly, call it <tt>ref-spades.pdf</tt> 
** What are vertical gray lines in the dotplot?
** Is any contig aligning to multiple places in the reference? To how many places?

* (c) Use analogous commands to create dotplot of reference to itself, call it <tt>ref-ref.pdf</tt>
** However, in the minimap2 command add option <tt>-p 0</tt> to include also weaker self-alignments
** Do you see any self-alignments, showing repeated sequences in the reference? Does this agree with dotplot in part (b)?

* '''Submit'''  all three pdf files ref-miniasm2.pdf, ref-spades.pdf, ref-ref.pdf

==Task E: Align reads and assemblies to reference, visualize in igv==

* Finally, we will align all source reads as well as assemblies to the reference genome, then visualize alignment in igv tool
** Write '''commands''' and '''answers''' to your '''protocol'''
** '''Submit''' all four bam files <tt>ref-miseq.bam</tt>, <tt>ref-nanopore.bam</tt>, <tt>ref-spades.bam</tt>, <tt>ref-miniasm2.bam</tt>

* (a) Align illumina reads (miseq files) to reference sequence
<pre>
# align illumina reads to reference
# minimap produces sam file, samtools view converts to bam, samtools sort orders by coordinate
minimap2 -a -x sr --secondary=no -t 1 ref.fasta  miseq_R1.fastq.gz miseq_R2.fastq.gz | samtools view -S -b - |  samtools sort - ref-miseq
# index bam file for faster access
samtools index ref-miseq.bam
</pre>
* (b) Similarly align nanopore reads. but instead of -x sr use -x map-ont, call the result ref-nanopore.bam, ref-nanopore.bam.bai
* (c) Similarly align spades.fasta, but instead of -x sr use -x asm10, call the result ref-spades.bam
* (d) Similarly align miniasm2.fasta, but instead of -x sr use -x asm10, call the result ref-miniasm2.bam
* (e) Run igv viewer. '''Beware: It needs a lot of memory, do not keep open unnecessarily'''
** <tt>igv -g ref.fasta &</tt>
** Using Menu->File->Load from File open all bam four files
** Look at region ecoli-frag:224,000-244,000
*** How many spades contigs do you see aligning in this region?
** Look at region ecoli-frag:227,300-227,600
*** Try to comment what you see, how frequent are errors in individual assemblies and read sets?
* If you are unable to run igv from home, you can install it on your computer [https://software.broadinstitute.org/software/igv/] and download ref.fasta and all bam and .bam.bai files

=L06bin=
* [[#HW06bin]]
==Eukaryotic gene structure==
* Recall the Central dogma of molecular biology: the flow of genetic information from DNA to RNA to protein (gene expression)
* In eukaryotes, mRNA often undergoes splicing, where introns are removed and exons are joined together
* The very start and end of mRNA remain untranslated (UTR = untranslated region)
* The coding part of the gene starts with a start codon, contains a sequence of additional codons and ends with a stop codon. Codons can be interrupted by  introns.

[[Image:Dogma.png|center|thumb|450px|Gene expression in eukaryotes]]

==Computational gene finding==
* Input: DNA sequence (an assembled genome or a part of it)
* Output: positions of protein coding genes and their exons
* If we know the exact position of coding regions of a gene, we can use genetic code to predict the protein sequence encoded by it
* Gene finders use statistical features observed from known genes, such as typical sequence motifs near the start codons, stop codons and splice sites, typical codon frequences, typical exon and intron lengths etc.
* These statistical parameters need to be adjusted for each genome.
* We will use a gene finder called [http://bioinf.uni-greifswald.de/augustus/ Augustus]

==Gene expression==
* Not all genes undergo transcription and translation all the time and at the same level
* The processes of transcription and translation are regulated according to cell needs
* The term "gene expression" has two meanings
** the process of transcription and translation (synthesis of a gene product)
** the amount of mRNA or protein produced from a single gene (genes with high or low expression)

* RNA-seq technology can sequence mRNA extracted from a sample of cells
* We can aligned sequenced reads back to the genome
* The number of reads coming from a gene depends on its expression level (and on its length)
=HW06bin=
[[#L06bin]]

==Input files, submitting==
Copy files from /tasks/hw06bin/
<pre>
mkdir hw06
cd hw06
cp -iv /tasks/hw06bin/* .
</pre>

Files:
* ref.fasta is a 38kb piece of genome of the fungus [https://www.ncbi.nlm.nih.gov/genome?term=aspergillus%20fumigatus Aspergillus nidulans]
* rnaseq.fastq are RNA-seq reads from Illumina sequencer extracted from the [https://www.ncbi.nlm.nih.gov/sra/?term=SRR4048918 Short read archive]
* annot.gff is the reference gene annotation from the database (we will consider his as correct gene positions)

Submit the protocol and the required files to /submit/hw06bin

==Task A: Gene finding==

Run the Augustus gene finder with two versions of parameters:
* one trained specifically for A. nidulas genes
* one trained for the human genome, where genes have different statistical properties (for example, they are longer and have more introns)
<pre>
augustus --species=anidulans ref.fasta > augustus-anidulans.gtf
augustus --species=human ref.fasta > augustus-human.gtf
</pre>

* The results of gene finding are in the [http://mblab.wustl.edu/GTF22.html GTF format]. Rows starting with # are comments, each of the remaining rows describes some interval of the sequence. If the second column is CDS, it is a coding part of an exon. 
* The reference annotation annot.gff is in a similar format called [http://gmod.org/wiki/GFF3 GFF3].

Examine the files and try to find the answers to the following questions using command-line tools
* (a) How many exons are in each of the two gtf files? (Beware: simply using grep with pattern CDS may yield lines containing this string in a different column. You can use e.g. techniques from [[#Lbash]] and [[#HWbash]]).
* (b) How many genes are in each of the two gtf files? (The files contain rows with word gene in the second column, one for each gene)
* (c) How many exons and genes are in the annot.gff file?

Write the anwsers and commands to the '''protocol'''. '''Submit''' files augustus-anidulans.gtf and augustus-human.gtf.

==Task B: Aligning RNA-seq reads==

* Align RNA-seq reads to the genome
* We will use a specialized tool tophat, which can recognize introns
* The we will sort and index bam the file, similarly as in [[#HWbioinf1]]

<pre>
bowtie2-build ref.fasta ref.fasta
tophat2 -i 10 -I 10000 --max-multihits 1 --output-dir rnaseq ref.fasta rnaseq.fastq
samtools sort rnaseq/accepted_hits.bam rnaseq
samtools index rnaseq.bam
</pre>

In addition to the bam file, TopHat produced several other files in the rnaseq folder. Examine them to find out answers to the following questions (you can do it manually by looking at the the files, e.g. by less command):
* (a) How many reads were in the the fastq file? How many of them were successfully mapped?
* (b) How many introns ("junctions") were predicted? How many of them are supported by more than one read? (The 5th column of the corresponding file is the number of reads supporting a junction.)

Write anwsers to the '''protocol'''. '''Submit''' file rnaseq.bam.

==Task C: Visualizing in igv==

As before, run igv as follows:
<pre>
igv -g ref.fasta &
</pre>

* Open additional files using menu File -> Load from File 
** annot.gff, augustus-anidulans.gtf, augustus-human.gtf, rnaseq.bam
* Exons are shown as thicker boxes, introns are thinner. 
* For each of the following questions, select part of the sequence illustrating the answer and export figure using File->Save image
* You can check these images using command eog

Questions:
* (a) Create image illustrating differences between Augustus with human parameters and the reference annotation, save as a.png. Briefly describe the differences in words.
* (b) Find some differences between Augustus with A.nidulans parameters and the reference annotation. Store an illustrative figure as b.png. Which parameters have yielded a more accurate prediction?
* (c) Zoom in to one of the genes with high expression level and try to find spliced read alignments supporting the annotated intron boundaries. Store the image as c.png.

'''Submit''' files a.png, b.png, c.png. Write answers to your '''protocol'''.

=L07bin=
* [[#HW07bin]]

==Polymorphisms==
* Individuals within species differ slightly in their genomes
* Polymorphisms are genome variants which are relatively frequent in a population (e.g. at least 1%)
* [https://ghr.nlm.nih.gov/primer/genomicresearch/snp SNP]: single-nucleotide polymorphism (a polymorphism which is a single substitution)
* Recall that most human cells are diploid, with one set of chromosomes inherited from the mother and the other from the father
* At a particular location, a single human can thus have two different alleles (heterozygosity) or two copies of the same allele (homozygosity)

==Finding polymorphisms / genome variants==
* We compare sequencing reads coming from an individual to a reference genome of the species
* First we align them, as in [[#HWbioinf1]]
* Then we look for positions where a substantial fraction of reads does not agree with the reference (SNP-calling)

==Programs and file formats==
* For mapping, we will use [https://github.com/lh3/bwa bwa mem] (you can also try minimap2, as in [[#HWbioinf1]])
* For SNP calling, we will use [https://github.com/ekg/freebayes freebayes]
* For reads and read alignments, we will use fastq and bam files, as in the [[#Lbioinf1|previous lectures]]
* For storing found variants, we will use [http://www.internationalgenome.org/wiki/Analysis/vcf4.0/ VCF files]
* For storing genome intervals, we will use [https://genome.ucsc.edu/FAQ/FAQformat.html#format1 BED files]

==Human variants==
* For many human SNPs we already know something about their influence on phenotype and their prevalence in different parts of the world
* There are various databases, e.g. [https://www.ncbi.nlm.nih.gov/SNP/ dbSNP], [https://www.omim.org/ OMIM], or user-editable [https://www.snpedia.com/index.php/SNPedia SNPedia]

==UCSC genome browser==
* On-line tool similar to IGV
* http://genome-euro.ucsc.edu/
* Nice interface for browsing genomes, lot of data for some genomes (particularly human), but not all sequenced genomes represented

====Basics====
* on the front page, choose Genomes in the top blue menu bar
* select a genome and its version, optionally enter position or keyword, press submit
* on the browser screen top image shows chromosome map, selected region in red
* below a view of selected region and various track with information about this region
* for example some of the top tracks display genes (boxes are exons, lines are introns)
* tracks can be switched on and off and configured in the bottom part of the page
** different display levels, full contains all information but takes a lot of vertical space
* navigation at the top (move, zoom, etc.)
* various actions in the menu
* clicking at the browser figure allows you to get more information about a gene or other displayed item
* this week, we will need tracks GENCODE and dbSNP - check e.g. [http://genome-euro.ucsc.edu/cgi-bin/hgTracks?db=hg38&position=chr11%3A66546841-66563329 gene ACTN3] and within it SNP rs1815739 in exon15

====Blat====
* UCSC genome browser uses a fast but less sensitive BLAT (good for the same or very closely related species)
* Choose Tools->Blat in the top blue menu bar, enter DNA sequence below, search in the human genome
** What is the identity level for the top found match? What is its span in the genome? (Notice that other matches are much shorter)
** Using Details link in the left column you can see the alignment itself, Browser link takes you to the browser at the matching region
<pre>
AACCATGGGTATATACGACTCACTATAGGGGGATATCAGCTGGGATGGCAAATAATGATTTTATTTTGAC
TGATAGTGACCTGTTCGTTGCAACAAATTGATAAGCAATGCTTTCTTATAATGCCAACTTTGTACAAGAA
AGTTGGGCAGGTGTGTTTTTTGTCCTTCAGGTAGCCGAAGAGCATCTCCAGGCCCCCCTCCACCAGCTCC
GGCAGAGGCTTGGATAAAGGGTTGTGGGAAATGTGGAGCCCTTTGTCCATGGGATTCCAGGCGATCCTCA
CCAGTCTACACAGCAGGTGGAGTTCGCTCGGGAGGGTCTGGATGTCATTGTTGTTGAGGTTCAGCAGCTC
CAGGCTGGTGACCAGGCAAAGCGACCTCGGGAAGGAGTGGATGTTGTTGCCCTCTGCGATGAAGATCTGC
AGGCTGGCCAGGTGCTGGATGCTCTCAGCGATGTTTTCCAGGCGATTCGAGCCCACGTGCAAGAAAATCA
GTTCCTTCAGGGAGAACACACACATGGGGATGTGCGCGAAGAAGTTGTTGCTGAGGTTTAGCTTCCTCAG
TCTAGAGAGGTCGGCGAAGCATGCAGGGAGCTGGGACAGGCAGTTGTGCGACAAGCTCAGGACCTCCAGC
TTTCGGCACAAGCTCAGCTCGGCCGGCACCTCTGTCAGGCAGTTCATGTTGACAAACAGGACCTTGAGGC
ACTGTAGGAGGCTCACTTCTCTGGGCAGGCTCTTCAGGCGGTTCCCGCACAAGTTCAGGACCACGATCCG
GGTCAGTTTCCCCACCTCGGGGAGGGAGAACCCCGGAGCTGGTTGTGAGACAAATTGAGTTTCTGGACCC
CCGAAAAGCCCCCACAAAAAGCCG
</pre>
=HW07bin=
[[#L07bin]]

==Input files, submitting==
Copy files from /tasks/hw07bin/
<pre>
mkdir hw07
cd hw07
cp -iv /tasks/hw07bin/* .
</pre>

Files:
* humanChr7Region.fasta is a 7kb piece of the human chromosome 7
* motherChr7Region.fastq is a sample of reads from an anonymous donor known as NA12878, these reads come from region in humanChr7Region.fasta
* fatherChr12.vcf and motherChr12.vcf are single-nucleotide variants in chr12 obtained by sequencing two individuals NA12877, NA12878 (these come from a larger [https://www.coriell.org/0/Sections/Collections/NIGMS/CEPHFamiliesDetail.aspx?PgId=441&fam=1463& family])

Submit the protocol and the required files to /submit/hw07bin

==Task A: read mapping and SNP calling==

Align reads to reference:
<pre>
bwa index humanChr7Region.fasta
bwa mem humanChr7Region.fasta  motherChr7Region.fastq | samtools view -S -b - |  samtools sort - motherChr7Region
samtools index motherChr7Region.bam
</pre>

Call SNPs:
<pre>
freebayes -f humanChr7Region.fasta --min-alternate-count 10 motherChr7Region.bam >motherChr7Region.vcf
</pre>

Run igv, use humanChr7Region.fasta as genome, open motherChr7Region.bam and motherChr7Region.vcf. Looking at the aligned reads and the vcf file, '''answer''' the following questions in protocol:
* (a) How many variants were found in the vcf file?
* (b) How many variants are heterozygous and how many are homozygous?
* (c) Are all variants single-nucleotide variants or do you also see some insertions/deletions (indels)?
Also export overall view of the whole region from igv to file motherChr7Region.png.

'''Submit''' the following files:
* motherChr7Region.png, motherChr7Region.bam, motherChr7Region.vcf

==Task B: UCSC browser==
* (a) Where is sequence from regionChr7.fasta located in the browser?
** Go to http://genome-euro.ucsc.edu/, From the blue menu, select Tools->Blat
** Check that blat uses Human, hg38 assembly
** Open regionChr7.fasta in a graphical editor (e.g. gedit), sleect all, paste to BLAT window, run BLAT 
** In the table of results, the best result should have identiy close to 100% and span close to 7kb
** For this best result, click on link named Browser
** Report which chromosome and which region you get

* (b) Which gene is located in this region?
** Once you are int he browser, press Default tracks button
** Track named GENCODE contains known genes, shown as rectangles (exanos) connected by lines (introns). Short gene names are next to them.
** Report the name of the gene in the region

* (c) In which tissue is this gene most highly expressed? What is the function of this gene?
** When you click on the gene (possibly twice), you get an information page which starts with a summary of the known function of this gene. Copy the first sentence to your protocol. 
** Further down on the gene information page you see RNA-Seq Expression Data (colorful boxplots). Find out which tissues have the highest signal.
* (d) Which SNPs are located in this gene? Which trait do they inflence?
** You can see SNPs in the Common SNPs(151) track, but their IDs appear only after switching this track to pack mode. You can click on each SNPs to see more information and to copy their ID to your protocol.
** Information page of the gene (part c) also describes function of various alleles of this gene (see e.g. part POLYMORPHISM).
** You can also find information about individual SNPs by looking for them by their ID in [https://www.snpedia.com/index.php/SNPedia SNPedia] (not required in this task)


<!--
* Default tracks
* Which gene (GENCODE)
* Which tissues (Gene express track)
* Switch on SNPedia track to "Pack", click on one of the SNPs within the gene, read the text - which trait do these SNPs influence?
-->

==Task C: Examining larger vcf files==
In this task, we will look at motherChr12.vcf and fatherChr12.vcf files and compute various statistics. You can use command-line tools, such as grep, wc, sort, uniq and Perl one-liners (as in [[#Lbash]]), you write small scripts in Perl or Python (as in [[#Lperl]] and [[#Lpython]]). 
* Write all used commands to your protocol
* If you write any scripts, submit them as well.

Questions:
* (a) How many SNPs are in each file?
** This can be found easily by wc, only make sure to exclude lines with comments
* (b) How many heterozygous SNPs are in each file?
** The last column contains 1|1 for homozygous and either 0|1 or 1|0 for heterozygous SNPs
** Character | has special meaning on command line and in grep patterns, make sure to place it in ' ' and possibly escape it with \
* (c) How many SNP positions are shared between the two files?
** The second column of each file lists the position. We want to compute the size of intersection of the set of positions in motherChr12.vcf and fatherChr12.vcf files
** You can e.g. create temporary files containing only positions from the two files and sort them alphabetically. Then you can find the intersection using [http://www.gnu.org/software/coreutils/manual/html_node/comm-invocation.html comm] command with options -1 -2. Alternatively, you can store positions as keys in a hash table (dictionary) in a Perl or Python script.
* (d) List the 5 most frequent pairs of reference/alternate allele in motherChr12.vcf and their frequencies. Do they correspond to transitions or transversions?
** Fourth column contains the reference value, fifth column the alternate value. For example, the first SNP in motherChr12.vcf has a pair C,A. 
** For each possible pair of nucleotides, find how many times it occurs in the motherChr12.vcf
** For example, pair C,A occurs 6894 times
** Then sort the pairs by their frequencies and report 5 most frequent pairs
** Mutations can be classified as transitions and transversions. Transitions change purine to purine or pyrimidine to pyrimidine, transversion change a purine to pyrimidine or vice versa. For example, pair C,A is a transversion changing pyrimidine C to purine A. Which of these most frequent pairs correspond to transitions and which to transversions? 
** To count pairs without writing scripts, you can create a temporary file containing only columns 4 and 5 (without comments), and then use commands sort and uniq to count each pair. 
* (e) Which parts of the chromosome have the highest and lowest number of SNPs in motherChr12.vcf?
** First create a list of windows of size 100kb covering the whole chromosome 12 using these two commands:
*** <tt>perl -le 'print "chr12\t133275309"' > humanChr12.size</tt>
*** <tt>bedtools makewindows -g humanChr12.size -w 100000 -i srcwinnum > humanChr12-windows.bed</tt>
** Then count SNPs in each window using this command:
*** <tt>bedtools coverage -a  humanChr12-windows.bed -b motherChr12.vcf > motherChr12-windows.tab</tt>
** Find out which column of the resulting file contains the number of SNPs per window, e.g. by reading the documentation obtained by command <tt>bedtools coverage -h</tt>
** Sort according to the column with SNP number, look at the first and last line of the sorted file
** For checking: the second highest count is 387 in window with coordinates 20,800,000-20,900,000
=L08=
[[#HW08]]

Program for today: basics of R (applied to biology examples)
* very short intro as a lecture
* tutorial as HW: read a bit of text, try some commands, extend/modify them as requested

In this course we cover several languages popular for scripting in bioinformatics: Perl, Python, R
* their capabilities overlap, many extensions emulate strengths of one in another
* choose a language based on your preference, level of knowledge, existing code for the task, rest of the team
* quickly learn a new language if needed
* also possibly combine, e.g. preprocess data in Perl or Python, then run statistical analyses in R, automate entire pipeline with bash or make

==Introduction==
* [http://www.r-project.org/ R] is an open-source system for statistical computing and data visualization
* Programming language, command-line interface
* Many built-in functions, additional libraries
** For example http://bioconductor.org/ for bioinformatics
* We will concentrate on useful commands rather than language features

==Working in R==
* Run command R, type commands in command-line interface
** supports history of commands (arrows, up and down, Ctrl-R) and completing command names with tab key 
<pre>
> 1+2
[1] 3
</pre>
* Write a script to file, run it from command-line: <tt>R --vanilla --slave < file.R</tt>
* Use <tt>rstudio</tt> to open a graphics IDE [https://www.rstudio.com/products/RStudio/]
** Windows with editor of R scripts, console, variables, plots
** Ctrl-Enter in editor executes current command in console
<pre>
x=c(1:10)
plot(x,x*x)
</pre>
* <tt>? plot</tt> displays help for plot command

Suggested workflow
* work interactively in Rstudio or on command line, try various options 
* select useful commands, store in a script
* run script automatically on new data/new versions, potentially as a part of a bigger pipeline

==Additional information==
* [http://cran.r-project.org/doc/manuals/R-intro.html Official tutorial]
* [http://cran.r-project.org/doc/contrib/Seefeld_StatsRBio.pdf Seefeld, Linder: Statistics Using R with Biological Examples (pdf book)]
* [http://www.burns-stat.com/pages/Tutor/R_inferno.pdf Patrick Burns: The R Inferno] (intricacies of the language) 
* [https://www.r-project.org/doc/bib/R-books.html Other books]

==Gene expression data==
* Gene expression: DNA->mRNA->protein
* Level of gene expression: Extract mRNA from a cell, measure amounts of mRNA
* Technologies: microarray, RNA-seq
Gene expression data
* Rows: genes
* Columns: experiments (e.g. different conditions or different individuals)
* Each value is expression of a gene, i.e. relative amount of mRNA for this gene in the sample

We will use microarray data for yeast:
* Strassburg, Katrin, et al. "Dynamic transcriptional and metabolic responses in yeast adapting to temperature stress." Omics: a journal of integrative biology 14.3 (2010): 249-259. [http://online.liebertpub.com/doi/full/10.1089/omi.2009.0107]
* Downloaded from GEO database [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE15352]
* Data already preprocessed: normalization, log2, etc
* We have selected only cold conditions, genes with absolute change at least 1
* Data: 2738 genes, 8 experiments in a time series, yeast moved from normal temperature 28 degrees C to cold conditions 10 degrees C, samples taken after 0min, 15min, 30min, 1h, 2h, 4h, 8h, 24h in cold
=HW08=
[[#L08]]

==Submitting==
In this homework, try to read text, execute given commands, potentially trying some small modifications.
* Then do tasks A-D, submit required files (3x .png)
* In your protocol, enter commands used in tasks A-D, with explanatory comments in more complicated situations
* In task B also enter required output to protocol
* Protocol template in /tasks/hw08/protocol.txt

==First steps==
* Type a command, R writes the answer, e.g.:
<pre>
> 1+2
[1] 3
</pre>
* We can store values in variables and use them later on
<pre>
> # The size of the sequenced portion of cow's genome, in millions of base pairs
> Cow_genome_size <- 2290
> Cow_genome_size
[1] 2290
> Cow_chromosome_pairs <- 30
> Cow_avg_chrom <- Cow_genome_size / Cow_chromosome_pairs
> Cow_avg_chrom
[1] 76.33333
</pre>
Surprises:
* dots are used as parts of id's, e.g. read.table is name of a single function (not method for object read)
* assignment via <- or = 
** careful: a<-3 is an assignment, a < -3 is a comparison
* vectors etc are indexed from 1, not from 0

==Vectors, basic plots==
* Vector is a sequence of values of the same type (all are numbers or all are strings or all are booleans)
<pre>
# Vector can be created from a list of numbers by function named c
a <- c(1,2,4)
a
# prints [1] 1 2 4

# c also concatenates vectors
c(a,a)
# prints [1] 1 2 4 1 2 4

# Vector of two strings 
b <- c("hello", "world")

# Create a vector of numbers 1..10
x <- 1:10
x
# prints [1]  1  2  3  4  5  6  7  8  9 10
</pre>

===Vector arithmetics===
* Operations applied to each member of the vector
<pre>
x <- 1:10
# Square each number in vector x
x*x
# prints [1]   1   4   9  16  25  36  49  64  81 100

# New vector y: logarithm of a number in x squared
y <- log(x*x)
y
# prints [1] 0.000000 1.386294 2.197225 2.772589 3.218876 3.583519 3.891820 4.158883
# [9] 4.394449 4.605170

# Draw graph of function log(x*x) for x=1..10
plot(x,y)
# The same graph but use lines instead of dots
plot(x,y,type="l")

# Addressing elements of a vector: positions start at 1
# Second element of the vector 
y[2]
# prints [1] 1.386294

# Which elements of the vector satisfy certain condition? (vector of logical values)
y>3
# prints [1] FALSE FALSE FALSE FALSE  TRUE  TRUE  TRUE  TRUE  TRUE  TRUE

# write only those elements from y that satisfy the condition
y[y>3]
# prints [1] 3.218876 3.583519 3.891820 4.158883 4.394449 4.605170

# we can also write values of x such that values of y satisfy the condition...
x[y>3]
# prints [1]  5  6  7  8  9 10
</pre>

* Alternative plotting facilities: [http://ggplot2.org/ ggplot2 library], [https://cran.r-project.org/web/packages/lattice/index.html lattice library]

===Task A===
* Create a plot of the '''binary logarithm''' with dots in the graph more densely spaced (from 0.1 to 10 with step 0.1)
* Store it in file <tt>log.png</tt> and '''submit''' this file
* Hints:
** Create x and y by vector arithmetics
** To compute binary logarithm check help <tt>? log</tt>
** Before running plot, use command <tt>png("log.png")</tt> to store the result, afterwards call <tt>dev.off()</tt> to close the file (in rstudio you can also export plots manually)

==Data frames and simple statistics==
* Data frame: a table similar to spreadsheet, each column is a vector, all are of the same length
* We will use a table with the following columns:
** The size of a genome, in millions of nucleotides
** Number of chromosome pairs
** GC content
** Taxonomic group mammal or fish
* Stored in CSV format, columns separated by tabs.
* Data: Han et al Genome Biology 2008 [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2441465/] 
<pre>
Species    Size    Chrom   GC      Group
Human      2850    23      40.9    mammal
Chimpanzee 2750    24      40.7    mammal
Macaque    2650    21      40.7    mammal
Mouse      2480    20      41.7    mammal
...
Tetraodon   187    21      45.9    fish
...
</pre>

<pre>
# reading a frame from file
a<-read.table("/tasks/hw08/genomes.csv", header = TRUE, sep = "\t");
# column with name size
a$Size

# Average chromosome length: divide size by the number of chromosomes
a$Size/a$Chrom

# Add average chromosome length as a new column to frame a
a<-cbind(a,AvgChrom=a$Size/a$Chrom)

# Scatter plot of average chromosome length vs GC content
plot(a$AvgChrom, a$GC)

# Compactly display structure of a 
# (good for checking that import worked etc)
str(a)

# display mean, median, etc. of each column
summary(a);

# average genome size
mean(a$Size)
# average genome size for mammals
mean(a$Size[a$Group=="mammal"])
# Standard deviation
sd(a$Size)

# Histogram of genome sizes
hist(a$Size)
</pre>

===Task B===
* Divide frame <tt>a</tt> to two frames, one for mammals, one for fish. Hint:
** Try command <tt>a[c(1,2,3),]</tt>. What is it doing?
** Try command <tt>a$Group=="mammal"</tt>.
** Combine these two commands to get rows for all mammals and store the frame in a new variable, then repeat for fish
** Use a general approach which does not depend on the exact number and ordering of rows in the table.

* Run the command <tt>summary</tt> separately for mammals and for fish. Which of their characteristics are different?
** '''Write''' output and your conclusion to the protocol

===Task C===
* Draw a graph comparing genome size vs GC content; use different colors for points representing mammals and fish
** '''Submit''' the plot in file <tt>genomes.png</tt>
** To draw the graph, you can use one of the options below, or find yet another way
** Option 1: first draw mammals with one color, then add fish in another color
*** Color of points can be changed by: <tt>plot(1:10,1:10, col="red")</tt>
*** After plot command you can add more points to the same graph by command <tt>points</tt>, which can be used similarly as <tt>plot</tt>
*** Warning: command <tt>points</tt> does not change the ranges of x and y axes. You have to set these manually so that points from both groups are visible. You can do this using options <tt>xlim</tt> and <tt>ylim</tt>, e.g. <tt>plot(x,y, col="red", xlim=c(1,100), ylim=c(1,100))</tt>
** Option 2: plot both mammals and fish in one plot command, and give it a vector of colors, one for each point
*** <tt>plot(1:10,1:10,col=c(rep("red",5),rep("blue",5)))</tt> will plot the first 5 points red and the last 5 points blue

* Bonus task: add a legend to the plot, showing which color is mammal and which is fish

==Expression data and clustering==

Data here is bigger, better to use plain R rather than rstudio (limited server CPU/memory)

<pre>
# Read gene expression data table
a <- read.table("/tasks/hw08/microarray.csv", header = TRUE, sep = "\t", row.names=1)
# Visual check of the first row
a[1,]
# plot starting point vs. situation after 1 hour
plot(a$cold_0min,a$cold_1h)
# to better see density in dense clouds of points, use this plot
smoothScatter(a$cold_15min, a$cold_1h)
# outliers away from diagonal in the plot above are most strongly differentially expressed genes
# these are more easy to see in MA plot:
# x-axis: average expression in the two conditions
# y-axis: difference between values (they are log-scale, so difference 1 means 2-fold)
smoothScatter((a$cold_15min+a$cold_1h)/2, a$cold_15min-a$cold_1h)
</pre>

Clustering is a wide group of methods that split data points into groups with similar properties
* We will group together genes that have a similar reaction to cold, i.e. their rows in gene expression data matrix have similar values
We will consider two simple clustering methods
* K means clustering splits points (genes) into ''k'' clusters, where ''k'' is a parameter given by the user. It finds a center of each cluster and tries to minimize the sum of distances from individual points to the center of their cluster. Note that this algorithm is randomized so you will get different clusters each time.
* Hierarchical clustering puts all data points (genes) to a hierarchy so that smallest subtrees of the hierarchy are the most closely related groups of points and these are connected to bigger and more loosely related groups.

[[Image:HW08-heatmap.png|thumb|200px|right|Example of a heatmap]]
<pre>
# Heatmap: creates hierarchical clustering of rows 
# then shows every value in the table using color ranging from red (lowest) to white (highest)
# Computation may take some time
heatmap(as.matrix(a),Colv=NA)
# Previous heatmap normalized each row, the next one uses data as they are:
heatmap(as.matrix(a),Colv=NA,scale="none")
</pre>

<pre>
# k means clustering to 7 clusters
k = 7
cl <- kmeans(a,k)
# each gene has assigned a cluster (number between 1 and k)
cl$cluster
# draw only cluster number 3 out of k
heatmap(as.matrix(a[cl$cluster==3,]),Rowv=NA, Colv=NA)

# reorder genes in the table according to cluster
heatmap(as.matrix(a[order(cl$cluster),]),Rowv=NA, Colv=NA)

# compare overall column means with column means in cluster 3
# function apply uses mean on every column (or row if 2 changed to 1)
apply(a,2,mean)
# now means within cluster
apply(a[cl$cluster==3,],2,mean)

# clusters have centers which are also computed as means
# so this is the same as previous command
cl$centers[3,]
</pre>

===Task D===
[[Image:HW08-clusters.png|thumb|200px|right|Example of a required plot]]
* Draw a plot in which x-axis is time and y-axis is the expression level and the center of each cluster is shown as a line
** use command <tt>matplot(x,y,type="l")</tt> which gets two matrices x and y and plots columns of x vs columns of y
** <tt>matplot(,y,type="l")</tt> will use numbers 1,2,3... as columns of the missing matrix x
** create y from <tt>cl$centers</tt> by applying function <tt>t</tt> (transpose)
** to create an appropriate matrix x, create a vector of times for individual experiments in minutes or hours (do it manually, no need to parse column names automatically)
** using functions <tt>rep</tt> and <tt>matrix</tt> you can create a matrix x in which this vector is used as every column
** then run <tt>matplot(x,y,type="l")</tt>
** since time points are not evenly spaced, it would be better to use logscale: <tt>matplot(x,y,type="l",log="x")</tt>
** to avoid log(0), change the first timepoint from 0min to 1min
* Submit file '''clusters.png''' with your final plot
=L09=
[[#HW09]]

Topic of this lecture are statistical tests in R.
* Beginners in statistics: listen to lecture, then do tasks A, B, C
* If you know basics of statistical tests, do tasks B, C, D
* More information on this topic in [https://sluzby.fmph.uniba.sk/infolist/sk/1-EFM-340_13.html 1-EFM-340 Počítačová štatistika]

==Introduction to statistical tests: sign test==
* [https://en.wikipedia.org/wiki/Sign_test]
* Two friends ''A'' and ''B'' have played their favourite game ''n''=10 times, ''A'' has won 6 times and ''B'' has won 4 times. 
* ''A'' claims that he is a better player, ''B'' claims that such a result could easily happen by chance if they were equally good players.
* Hypothesis of player ''B'' is called ''null hypothesis'' that the pattern we see (''A'' won more often than ''B'') is simply a result of chance
* Null hypothesis reformulated: we toss coin ''n'' times and compute value ''X'': the number of times we see head. The tosses are independent and each toss has equal probability of being head or tail
* Similar situation: comparing programs A and B on several inputs, counting how many times is program A better than B.
<pre>
# simulation in R: generate 10 psedorandom bits
# (1=player A won)
sample(c(0,1), 10, replace = TRUE)
# result e.g. 0 0 0 0 1 0 1 1 0 0

# directly compute random variable X, i.e. sum of bits
sum(sample(c(0,1), 10, replace = TRUE))
# result e.g. 5

# we define a function which will m times repeat 
# the coin tossing experiment with n tosses 
# and returns a vector with m values of random variable X
experiment <- function(m, n) {
  x = rep(0, m)     # create vector with m zeroes
  for(i in 1:m) {   # for loop through m experiments
    x[i] = sum(sample(c(0,1), n, replace = TRUE)) 
  }
  return(x)         # return array of values     
}
# call the function for m=20 experiments, each with n tosses
experiment(20,10)
# result e.g.  4 5 3 6 2 3 5 5 3 4 5 5 6 6 6 5 6 6 6 4
# draw histograms for 20 experiments and 1000 experiments
png("hist10.png")  # open png file
par(mfrow=c(2,1))  # matrix of plots with 2 rows and 1 column
hist(experiment(20,10))
hist(experiment(1000,10))
dev.off() # finish writing to file
</pre>
* It is easy to realize that we get [https://en.wikipedia.org/wiki/Binomial_distribution binomial distribution] (binomické rozdelenie)
* <math>Pr(X=k) = {n \choose k} 2^{-n}</math>
* ''P-value'' of the test is the probability that simply by chance we would get ''k'' the same or more extreme than in our data.
* In other words, what is the probability that in 10 tosses we see head 6 times or more (one sided test)
* <math>\sum_{j=k}^n {n \choose k} 2^{-n}</math>
* If the p-value is very small, say smaller than 0.01, we reject the null hypothesis and assume that player ''A'' is in fact better than ''B''

<pre>
# computing the probability that we get exactly 6 heads in 10 tosses
dbinom(6, 10, 0.5) # result 0.2050781
# we get the same as our formula above:
7*8*9*10/(2*3*4*(2^10)) # result 0.2050781

# entire probability distribution: probabilities 0..10 heads in 10 tosses
dbinom(0:10, 10, 0.5)
# [1] 0.0009765625 0.0097656250 0.0439453125 0.1171875000 0.2050781250
# [6] 0.2460937500 0.2050781250 0.1171875000 0.0439453125 0.0097656250
# [11] 0.0009765625

#we can also plot the distribution
plot(0:10, dbinom(0:10, 10, 0.5))
barplot(dbinom(0:10,10,0.5))

#our p-value is sum for 6,7,8,9,10
sum(dbinom(6:10,10,0.5))
# result: 0.3769531
# so results this "extreme" are not rare by chance,
# they happen in about 38% of cases

# R can compute the sum for us using pbinom 
# this considers all values greater than 5
pbinom(5, 10, 0.5, lower.tail=FALSE)
# result again 0.3769531

# if probability is too small, use log of it
pbinom(9999, 10000, 0.5, lower.tail=FALSE, log.p = TRUE)
# [1] -6931.472
# the probability of getting 10000x head is exp(-6931.472) = 2^{-100000}

# generating numbers from binomial distribution 
# - similarly to our function experiment
rbinom(20, 10, 0.5)
# [1] 4 4 8 2 6 6 3 5 5 5 5 6 6 2 7 6 4 6 6 5

# running the test
binom.test(6, 10, p = 0.5, alternative="greater")
#
#        Exact binomial test
#
# data:  6 and 10
# number of successes = 6, number of trials = 10, p-value = 0.377
# alternative hypothesis: true probability of success is greater than 0.5
# 95 percent confidence interval:
# 0.3035372 1.0000000
# sample estimates:
# probability of success
#                   0.6

# to only get p-value run
binom.test(6, 10, p = 0.5, alternative="greater")$p.value
# result 0.3769531
</pre>

==Comparing two sets of values: Welch's t-test==
* Let us now consider two sets of values drawn from two [https://en.wikipedia.org/wiki/Normal_distribution normal distributions] with unknown means and variances
* The null hypothesis of the [https://en.wikipedia.org/wiki/Welch%27s_t-test Welch's t-test] is that the two distributions have equal means
* The test computes test statistics (in R for vectors x1, x2):
** <tt>(mean(x1)-mean(x2))/sqrt(var(x1)/length(x1)+var(x2)/length(x2))</tt>
* This test statistics is approximately distributed according to [https://en.wikipedia.org/wiki/Student%27s_t-distribution Student's t-distribution] with the degree of freedom obtained by 
<pre>
n1=length(x1)
n2=length(x2)
(var(x1)/n1+var(x2)/n2)**2/(var(x1)**2/((n1-1)*n1*n1)+var(x2)**2/((n2-1)*n2*n2))
</pre>
* Luckily R will compute the test for us simply by calling t.test
<pre>
x1 = rnorm(6, 2, 1)
# 2.70110750  3.45304366 -0.02696629  2.86020145  2.37496993  2.27073550

x2 = rnorm(4, 3, 0.5)
# 3.258643 3.731206 2.868478 2.239788
> t.test(x1,x2)
# t = -1.2898, df = 7.774, p-value = 0.2341
# alternative hypothesis: true difference in means is not equal to 0
# means 2.272182  3.024529

x2 = rnorm(4, 5, 0.5)
# 4.882395 4.423485 4.646700 4.515626
t.test(x1,x2)
# t = -4.684, df = 5.405, p-value = 0.004435
# means 2.272182  4.617051

# to get only p-value, run 
t.test(x1,x2)$p.value
</pre>

We will apply Welch's t-test to microarray data
* Data from GEO database [http://www.ncbi.nlm.nih.gov/sites/GDSbrowser?acc=GDS2925], publication [http://femsyr.oxfordjournals.org/content/7/6/819.abstract]
* Abbott et al 2007: Generic and specific transcriptional responses to different weak organic acids in anaerobic chemostat cultures of Saccharomyces cerevisiae 
* gene expression measurements under 5 conditions:
** reference: yeast grown in normal environment
** 4 different acids added so that cells grow 50% slower (acetic, propionic, sorbic, benzoic)
* from each condition (reference and each acid) we have 3 replicates
* together our table has 15 columns (3 replicates from 5 conditions)
* 6398 rows (genes)
* We will test statistical difference between the reference condition and one of the acids (3 numbers vs other 3 numbers)
* See Task B in [[#HW09]]

==Multiple testing correction==

* When we run t-tests on the reference vs. acetic acid on all 6398 genes, we get 118 genes with p-value<=0.01
* Purely by chance this would happen in 1% of cases (from definition of p-value)
* So purely by chance we would expect to get about 64 genes with p-value<=0.01
* So perhaps roughly half of our detected genes (maybe less, maybe more) are false positives
* Sometimes false positives may even overwhelm results
* Multiple testing correction tries to limit the number of false positives among results of multiple statistical tests 
* Many different methods
* The simplest one is [https://en.wikipedia.org/wiki/Bonferroni_correction Bonferroni correction], where the threshold on p-value is divided by the number of tested genes, so instead of 0.01 we use 0.01/6398 =  1.56e-6
* This way the expected overall number of false positives in the whole set is 0.01 and so the probability of getting even a single false positive is also at most 0.01 (by Markov inequality)
* We could instead multiply all p-values by the number of tests and apply the original threshold 0.01 - such artificially modified p-values are called corrected
* After Bonferroni correction we get only 1 significant gene
<pre>
# the results of p-tests are in vector pa of length 6398
# manually multiply p-values by length(pa), count those that have value <=0.01
sum(pa * length(pa) < 0.01)
# in R you can use p.adjust form multiple testing correction
pa.adjusted = p.adjust(pa, method ="bonferroni")
# this is equivalent to multiplying by the length and using 1 if the result > 1
pa.adjusted = pmin(pa*length(pa),rep(1,length(pa)))

# there are less conservative multiple testing correction methods, e.g. Holm's method
# but in this case we get almost the same results
pa.adjusted2 = p.adjust(pa, method ="holm")
</pre>
* Other frequently used correction is [https://en.wikipedia.org/wiki/False_discovery_rate false discovery rate (FDR)], which is less strict and controls the overall proportion of false positives among results
=HW09=
[[#L09]]
* Do either tasks A,B,C (beginners) or B,C,D (more advanced). If you really want, you can do all four for bonus credit.
* In your protocol write used R commands with brief comments on your approach.
* Submit required plots with filenames as specified.
* For each task also include results as required and a short discussion commenting the results/plots you have obtained. Is the value of interest increasing or decreasing with some parameter? Are the results as expected or surprising?
* Outline of protocol is in /tasks/hw09/protocol.txt

==Task A: sign test==

* Consider a situation in which players played ''n'' games, out of which a fraction of ''q'' were won by ''A'' (example in lecture corresponds to ''q=0.6'' and ''n=10'')
* Compute a table of p-values for ''n=10,20,...,90,100'' and for ''q=0.6, 0.7, 0.8, 0.9''
* Plot the table using matplot (''n'' is x-axis, one line for each value of ''q'')
* '''Submit''' the plot in <tt>sign.png</tt>
* '''Discuss''' the values you have seen in the plot / table

Outline of the code:
<pre>
# create vector rows with values 10,20,...,100
rows=(1:10)*10
# create vector columns with required values of q
columns=c(0.6, 0.7, 0.8, 0.9)
# create empty matrix of pvalues 
pvalues = matrix(0,length(rows),length(columns))
# TODO: fill in matrix pvalues using binom.test

# set names of rows and columns
rownames(pvalues)=rows
colnames(pvalues)=columns
# careful: pvalues[10,] is now 10th row, i.e. value for n=100, 
#          pvalues["10",] is the first row, i.e. value for n=10

# check that for n=10 and q=0.6 you get p-value 0.3769531
pvalues["10","0.6"]

# create x-axis matrix (as in HW08, part D)
x=matrix(rep(rows,length(columns)),nrow=length(rows))
# matplot command
png("sign.png")
matplot(x,pvalues,type="l",col=c(1:length(columns)),lty=1)
legend("topright",legend=columns,col=c(1:length(columns)),lty=1)
dev.off()
</pre>

==Task B: Welch's t-test on microarray data==

* Read table with microarray data, transform it to log scale, then work with table ''a'':
<pre>
input=read.table("/tasks/hw09/acids.tsv", header=TRUE, row.names=1)
a = log(input)
</pre>
* Columns 1,2,3 are reference, columns 4,5,6 acetic acid, 7,8,9 benzoate, 10,11,12 propionate, and 13,14,15 sorbate
* Write a function <tt>my.test</tt> which will take as arguments table ''a'' and 2 lists of columns (e.g. 1:3 and 4:6) and will run for each row of the table Welch's t-test of the first set of columns vs the second set. It will return the resulting vector of p-values
* For example by calling <tt>pa <- my.test(a, 1:3, 4:6)</tt> we will compute p-values for differences between reference and acetic acid (computation may take some time)
* The first 5 values of pa should be
<pre>
> pa[1:5]
[1] 0.94898907 0.07179619 0.24797684 0.48204100 0.23177496
</pre>
* Run the test for all four acids
* '''Report''' how many genes were significant with p-value <= 0.01 for each acid
** See [[#HW08#Vector_arithmetics|Vector arithmetics in HW08]]
** You can count TRUE items in a vector of booleans by sum, e.g. <tt>sum(TRUE,FALSE,TRUE)</tt> is 2
* '''Report''' how many genes are significant for both acetic and benzoate acids? (logical and is written as <tt>&</tt>)

==Task C: multiple testing correction==

Run the following snippet of code, which works on the vector of p-values <tt>pa</tt> obtained for acetate in task B
<pre>
# adjusts vectors of p-vales from tasks B for using Bonferroni correction
pa.adjusted = p.adjust(pa, method ="bonferroni")
# add this adjusted vector to frame a
a <-  cbind(a, pa.adjusted)
# create permutation ordered by pa.adjusted
oa = order(pa.adjusted)
# select from table five rows with the lowest pa.adjusted (using vector oa)
# and display columns containing reference, acetate and adjusted p-value
a[oa[1:5],c(1:6,16)]
</pre>

You should get output like this:
<pre>
            ref1     ref2     ref3  acetate1   acetate2  acetate3 pa.adjusted
SUL1    7.581312 7.394985 7.412040 2.1633230 2.05412373 1.9169226 0.004793318
YMR244W 2.985682 2.975530 3.054001 0.3364722 0.33647224 0.1823216 0.188582576
DIP5    6.943991 7.147795 7.296955 0.6931472 0.09531018 0.5306283 0.253995075
YLR460C 5.620401 5.801212 5.502482 3.2425924 3.48431229 3.3843903 0.307639012
HXT4    2.821379 3.049273 2.772589 7.7893717 8.24446541 8.3041980 0.573813502
</pre>

Do the same procedure for benzoate p-values and '''report''' the result (in your table, report both p-values and expression levels for bezoate, not acetate). '''Comment''' the results for both acids.

==Task D: volcano plot, test on data generated from null hypothesis==
Draw a [https://en.wikipedia.org/wiki/Volcano_plot_(statistics) volcano plot] for the acetate data
* x-axis of this plot is the difference in the mean of reference and mean of acetate. 
** You can compute row means of a matrix by rowMeans.
* y-axis is -log10 of the p-value (use original p-values before multiple testing correction)
* You can quickly see genes which have low p-values (high on y-axis) and also big difference in mean expression between the two conditions (far from 0 on x-axis). You can also see if acetate increases or decreases expression of these genes.

Now create a simulated dataset sharing some features of the real data but observing the null hypothesis that the mean of reference and acetate are the same for each gene
* Compute vector ''m'' of means for columns 1:6 from matrix ''a''
* Compute vectors ''sr'' and ''sa'' of standard deviations for reference columns and for acetate columns respectively
** You can compute standard deviation for each row of a matrix by <tt>apply(some.matrix, 1, sd)</tt>
* For each i in 1:6398, create three samples from normal distribution with mean ''m[i]'' and standard deviation ''sr[i]'', and three samples with mean m[i] and deviation sa[i]
** Use function <tt>rnorm</tt>
* On the resulting matrix apply Welch's t-test and draw the volcano plot. 
* How many random genes have p-value <=0.01? Is it roughly what we would expect under the null hypothesis?

Draw histogram of p-values from the real data (reference vs acetate) and from random data. '''Describe''' how they differ. Is it what you would expect?
* use function <tt>hist</tt>

'''Submit''' plots <tt>volcano-real.png</tt>, <tt>volcano-random.png</tt>, <tt>hist-real.png</tt>, <tt>hist-random.png</tt>
(real for real expression data and random for generated data)
=L10=
Today we will work with AWS.
Please use credentials which were sent to you via email and follows steps in here (there is a cursor in each screen):
https://docs.google.com/presentation/d/1GBDErp5xhrV2zLF5kKdwnOAjtmDEFN0pw3RFval419s/edit#slide=id.p

The goal is to have '''.aws/credentials'' file in your homefolder.
Sometimes these credentials expire, follow same steps to refresh them (copy them again).

There is aws command line tools installed on vyuka machine (on your machine use '''pip install awscli''').

Now you need to do two things:

Check if you can download a file from our S3 datastore (think of S3 as some remote file storage).

'''aws s3 ls s3://idzbucket2''' - should give you a big list of files
'''aws s3 cp s3://idzbucket2/splitaa splitaa''' - should download a file to your machine
'''aws s3 cp s3://idzbucket2/splitaa -''' - will print file in your console (no need to do this).

Check if you can create a bucket (your storage area, pick your own name, must be globally unique):
'''aws s3 mb s3://mysuperawesomebucket'''

We will be using MapReduce in this session. (It is kind of outdated concept, but simple enough for us and runs out of box on AWS. 
If you ever want to use BigData in practice, try something more modern like Apache Beam. And avoid PySpark if you can.)

For tutorial on mapreduce check out [https://pythonhosted.org/mrjob/guides/concepts.html#mapreduce-and-apache-hadoop] or [https://www.tutorialspoint.com/map_reduce/map_reduce_introduction.htm].

You are given basic template with comments at '''tasks/example_job.py''' and you can run it locally as '''python3 example_job.py <input file> -o <output_dir>''' or in cloud as '''python3 example_job.py -r emr s3://idzbucket2 --num-core-instances 4 -o s3://<your bucket>/<some directory>''' (but for testing we recommend smaller sample '''python3 example_job.py -r emr s3://idzbucket2/splita* --num-core-instances 4 -o  s3://<your bucket>/<some directory>''').

You can download output using '''aws s3 ls s3://<your bucket>/<some directory>/''' and  '''aws s3 cp s3://<your bucket>/<some directory>/ . --recursive'''

If you want to watch progress:
Click on AWS Console button workbench (vocareum).
Set region (top right) to Oregon.
Click on services, then EMR.
Click on the job, which is running, then Steps, view logs, syslog.
=HW10=
Task 1:

Count number of occurences of each 4-mer in provided data.

Task 2:

Count number of pairs of reads which overlaps in exactly 30 bases (end of one read overlaps beginning of second read).
You can ignore reverse complement.
Hints: 

* Try counting pairs for each 30-mer first.
* You can yield something structured from Mapper (e.g. tuple).
* There is a two step mapreduce, which can help you with final summation: https://pythonhosted.org/mrjob/guides/writing-mrjobs.html


For both tasks, submit source code and the result, when run on whole dataset (s3://idzbucket2).
Code is expected to use MRJob framework presented in lecture.